Selective re-routing of prion protein to proteasomes and alteration of its vesicular secretion prevent PrP(Sc) formation

Conversion of the cellular prion protein (PrP(C)) into the abnormal scrapie isoform (PrP(Sc)) is the hallmark of prion diseases, which are fatal and transmissible neurodegenerative disorders. ER-retained anti-prion recombinant single-chain Fv fragments have been proved to be an effective tool for inhibition of PrP(C) trafficking to the cell surface and antagonize PrP(Sc) formation and infectivity. In the present study, we have generated the secreted version of 8H4 intrabody (Sec-8H4) in order to compel PrP(C) outside the cells. The stable expression of the Sec-8H4 intrabodies induces proteasome degradation of endogenous prion protein but does not influence its glycosylation profile and maturation. Moreover, we found a dramatic diverting of PrP(C) traffic from its vesicular secretion and, most importantly, a total inhibition of PrP(Sc) accumulation in NGF-differentiated Sec-8H4 PC12 cells. These results confirm that perturbing the intracellular traffic of endogenous PrP(C) is an effective strategy to inhibit PrP(Sc) accumulation and provide convincing evidences for application of intracellular antibodies in prion diseases.     

The tyrosine kinase inhibitor STI571 induces cellular clearance of PrPSc in prion-infected cells

The conversion of the cellular prion protein (PrP(c)) into pathologic PrP(Sc) and the accumulation of aggregated PrP(Sc) are hallmarks of prion diseases. A variety of experimental approaches to interfere with prion conversion have been reported. Our interest was whether interference with intracellular signaling events has an impact on this conversion process. We screened approximately 50 prototype inhibitors of specific signaling pathways in prion-infected cells for their capacity to affect prion conversion. The tyrosine kinase inhibitor STI571 was highly effective against PrP(Sc) propagation, with an IC(50) of < or =1 microM. STI571 cleared prion-infected cells in a time- and dose-dependent manner from PrP(Sc) without influencing biogenesis, localization, or biochemical features of PrP(c). Interestingly, this compound did not interfere with the de novo formation of PrP(Sc) but activated the lysosomal degradation of pre-existing PrP(Sc), lowering the half-life of PrP(Sc) from > or =24 h to <9 h. Our data indicate that among the kinases known to be inhibited by STI571, c-Abl is likely responsible for the observed anti-prion effect. Taken together, we demonstrate that treatment with STI571 strongly activates the lysosomal degradation of PrP(Sc) and that substances specifically interfering with cellular signaling pathways might represent a novel class of anti-prion compounds.     

PrPSc binding antibodies are potent inhibitors of prion replication in cell lines

Conversion of the cellular alpha-helical prion protein (PrP(C)) into a disease-associated isoform (PrP(Sc)) is central to the pathogenesis of prion diseases. Molecules targeting either normal or disease-associated isoforms may be of therapeutic interest, and the antibodies binding PrP(C) have been shown to inhibit prion accumulation in vitro. Here we investigate whether antibodies that additionally target disease-associated isoforms such as PrP(Sc) inhibit prion replication in ovine PrP-inducible scrapie-infected Rov cells. We conclude from these experiments that antibodies exclusively binding PrP(C) were relatively inefficient inhibitors of ScRov cell PrP(Sc) accumulation compared with antibodies that additionally targeted disease-associated PrP isoforms. Although the mechanism by which these monoclonal antibodies inhibit prion replication is unclear, some of the data suggest that antibodies might actively increase PrP(Sc) turnover. Thus antibodies that bind to both normal and disease-associated isoforms represent very promising anti-prion agents.     

Caspase-12 and endoplasmic reticulum stress mediate neurotoxicity of pathological prion protein

Prion diseases are characterized by accumulation of misfolded prion protein (PrP(Sc)), and neuronal death by apoptosis. Here we show that nanomolar concentrations of purified PrP(Sc) from mouse scrapie brain induce apoptosis of N2A neuroblastoma cells. PrP(Sc) toxicity was associated with an increase of intracellular calcium released from endoplasmic reticulum (ER) and up-regulation of several ER chaperones. Caspase-12 activation was detected in cells treated with PrP(Sc), and cellular death was inhibited by overexpression of a catalytic mutant of caspase-12 or an ER-targeted Bcl-2 chimeric protein. Scrapie-infected N2A cells were more susceptible to ER-stress and to PrP(Sc) toxicity than non-infected cells. In scrapie-infected mice a correlation between caspase-12 activation and neuronal loss was observed in histological and biochemical analyses of different brain areas. The extent of prion replication was closely correlated with the up-regulation of ER-stress chaperone proteins. Similar results were observed in humans affected with sporadic and variant Creutzfeldt-Jakob disease, implicating for the first time the caspase-12 dependent pathway in a neurodegenerative disease in vivo, and thus offering novel potential targets for the treatment of prion disorders.     

The role of rafts in the fibrillization and aggregation of prions

A key molecular event in prion diseases is the conversion of the prion protein (PrP) from its normal cellular form (PrP(C)) to the disease-specific form (PrP(Sc)). The transition from PrP(C) to PrP(Sc) involves a major conformational change, resulting in amorphous aggregates and/or fibrillar amyloid deposits. Here several lines of evidence implicating membranes in the conversion of PrP are reviewed with a particular emphasis on the role of lipid rafts in the conformational transition of prion proteins. New correlations between in vitro biophysical studies and findings from cell biology work on the role of rafts in prion conversion are highlighted and a mechanism for the role of rafts in prion conversion is proposed.     

Altered prion protein glycosylation in the aging mouse brain

The normal cellular prion protein (PrP(C)) is a glycoprotein with two highly conserved potential N-linked glycosylation sites. All prion diseases, whether inherited, infectious or sporadic, are believed to share the same pathogenic mechanism that is based on the conversion of the normal cellular prion protein (PrP(C)) to the pathogenic scrapie prion protein (PrP(Sc)). However, the clinical and histopathological presentations of prion diseases are heterogeneous, depending not only on the strains of PrP(Sc) but also on the mechanism of diseases, such as age-related sporadic vs. infectious prion diseases. Accumulated evidence suggests that N-linked glycans on PrP(C) are important in disease phenotype. A better understanding of the nature of the N-linked glycans on PrP(C) during the normal aging process may provide new insights into the roles that N-linked glycans play in the pathogenesis of prion diseases. By using a panel of 19 lectins in an antibody-lectin enzyme-linked immunosorbent assay (ELISA), we found that the lectin binding profiles of PrP(C) alter significantly during aging. There is an increasing prevalence of complex oligosaccharides on the aging PrP(C), which are features of PrP(Sc). Taken together, this study suggests a link between the glycosylation patterns on PrP(C) during aging and PrP(Sc).     

Evolving views in prion glycosylation: functional and pathological implications

Prion diseases form a group of neurodegenerative disorders with the unique feature of being transmissible. These diseases involve a pathogenic protein, called PrP(Sc) for the scrapie isoform of the cellular prion protein (PrP(C)) which is an abnormally-folded counterpart of PrP(C). Many questions remain unresolved concerning the function of PrP(C) and the mechanisms underlying prion replication, transmission and neurodegeneration. PrP(C) is a glycosyl-phosphatidylinositol-anchored glycoprotein expressed at the cell surface of neurons and other cell types. PrP(C) may be present as distinct isoforms depending on proteolytic processing (full length and truncated), topology(GPI-anchored, transmembrane or soluble) and glycosylation (non- mono- and di-glycosylated). The present review focuses on the implications of PrP(C) glycosylation as to the function of the normal protein, the cellular pathways of conversion into PrP(Sc), the diversity of prion strains and the related selective neuronal targeting.     

Long-time scale fluctuations of human prion protein determined by restrained MD simulations

Cellular prion protein (PrP(C)) has the ability to trigger transmissible lethal diseases after in vivo maturation into a toxic amyloidogenic misfolded form (PrP(Sc)). Here, we use hydrogen exchange protection factors in restrained molecular dynamics simulations to characterize long-time scale fluctuations in human PrP(C). We find that the regions of residues 138-141 and 183-192 form new β-strands in several exchange-competent structures. Moreover, these structural changes are associated with the disruption of native contacts that when tethered prevent fibril formation. Our findings illustrate the structural plasticity of PrP(C) and are valuable for understanding the conversion of PrP(C) to PrP(Sc).     

Disease-associated F198S mutation increases the propensity of the recombinant prion protein for conformational conversion to scrapie-like form

The critical step in the pathogenesis of transmissible spongiform encephalopathies (prion diseases) is the conversion of a cellular prion protein (PrP(c)) into a protease-resistant, beta-sheet rich form (PrP(Sc)). Although the disease transmission normally requires direct interaction between exogenous PrP(Sc) and endogenous PrP(C), the pathogenic process in hereditary prion diseases appears to develop spontaneously (i.e. not requiring infection with exogenous PrP(Sc)). To gain insight into the molecular basis of hereditary spongiform encephalopathies, we have characterized the biophysical properties of the recombinant human prion protein variant containing the mutation (Phe(198) --> Ser) associated with familial Gerstmann-Straussler-Scheinker disease. Compared with the wild-type protein, the F198S variant shows a dramatically increased propensity to self-associate into beta-sheet-rich oligomers. In a guanidine HCl-containing buffer, the transition of the F198S variant from a normal alpha-helical conformation into an oligomeric beta-sheet structure is about 50 times faster than that of the wild-type protein. Importantly, in contrast to the wild-type PrP, the mutant protein undergoes a spontaneous conversion to oligomeric beta-sheet structure even in the absence of guanidine HCl or any other denaturants. In addition to beta-sheet structure, the oligomeric form of the protein is characterized by partial resistance to proteinase K digestion, affinity for amyloid-specific dye, thioflavine T, and fibrillar morphology. The increased propensity of the F198S variant to undergo a conversion to a PrP(Sc)-like form correlates with a markedly decreased thermodynamic stability of the native alpha-helical conformer of the mutant protein. This correlation supports the notion that partially unfolded intermediates may be involved in conformational conversion of the prion protein.     

Experimental approaches to the interaction of the prion protein with nucleic acids and glycosaminoglycans: Modulators of the pathogenic conversion

The concept that transmissible spongiform encephalopathies (TSEs) are caused only by proteins has changed the traditional paradigm that disease transmission is due solely to an agent that carries genetic information. The central hypothesis for prion diseases proposes that the conversion of a cellular prion protein (PrP(C)) into a misfolded, β-sheet-rich isoform (PrP(Sc)) accounts for the development of (TSE). There is substantial evidence that the infectious material consists chiefly of a protein, PrP(Sc), with no genomic coding material, unlike a virus particle, which has both. However, prions seem to have other partners that chaperone their activities in converting the PrP(C) into the disease-causing isoform. Nucleic acids (NAs) and glycosaminoglycans (GAGs) are the most probable accomplices of prion conversion. Here, we review the recent experimental approaches that have been employed to characterize the interaction of prion proteins with nucleic acids and glycosaminoglycans. A PrP recognizes many nucleic acids and GAGs with high affinities, and this seems to be related to a pathophysiological role for this interaction. A PrP binds nucleic acids and GAGs with structural selectivity, and some PrP:NA complexes can become proteinase K-resistant, undergoing amyloid oligomerization and conversion to a β-sheet-rich structure. These results are consistent with the hypothesis that endogenous polyanions (such as NAs and GAGs) may accelerate the rate of prion disease progression by acting as scaffolds or lattices that mediate the interaction between PrP(C) and PrP(Sc) molecules. In addition to a still-possible hypothesis that nucleic acids and GAGs, especially those from the host, may modulate the conversion, the recent structural characterization of the complexes has raised the possibility of developing new diagnostic and therapeutic strategies.     

Human cellular prion protein hPrPC is sorted to the apical membrane of epithelial cells

Propagation of the scrapie isoform of the prion protein (PrP(Sc)) depends on the expression of endogenous cellular prion (PrP(C)). During oral infection, PrP(Sc) propagates, by conversion of the PrP(C) to PrP(Sc), from the gastrointestinal tract to the nervous system. Intestinal epithelium could serve as the primary site for PrP(C) conversion. To investigate PrP(C) sorting in epithelia cells, we have generated both a green fluorescent protein (EGFP) or hemagglutinin (HA) tagged human PrP(C) (hPrP(C)). Combined molecular, biochemical, and single living polarized cell imaging characterizations suggest that hPrP(C) is selectively targeted to the apical side of Madin-Darby canine kidney (MDCKII) and of intestinal epithelia (Caco2) cells.     

Mouse prion protein (PrP) segment 100 to 104 regulates conversion of PrP(C) to PrP(Sc) in prion-infected neuroblastoma cells

Prion diseases are characterized by the replicative propagation of disease-associated forms of prion protein (PrP(Sc); PrP refers to prion protein). The propagation is believed to proceed via two steps; the initial binding of the normal form of PrP (PrP(C)) to PrP(Sc) and the subsequent conversion of PrP(C) to PrP(Sc). We have explored the two-step model in prion-infected mouse neuroblastoma (ScN2a) cells by focusing on the mouse PrP (MoPrP) segment 92-GGTHNQWNKPSKPKTN-107, which is within a region previously suggested to be part of the binding interface or shown to differ in its accessibility to anti-PrP antibodies between PrP(C) and PrP(Sc). Exchanging the MoPrP segment with the corresponding chicken PrP segment (106-GGSYHNQKPWKPPKTN-121) revealed the necessity of MoPrP residues 99 to 104 for the chimeras to achieve the PrP(Sc) state, while segment 95 to 98 was replaceable with the chicken sequence. An alanine substitution at position 100, 102, 103, or 104 of MoPrP gave rise to nonconvertible mutants that associated with MoPrP(Sc) and interfered with the conversion of endogenous MoPrP(C). The interference was not evoked by a chimera (designated MCM2) in which MoPrP segment 95 to 104 was changed to the chicken sequence, though MCM2 associated with MoPrP(Sc). Incubation of the cells with a synthetic peptide composed of MoPrP residues 93 to 107 or alanine-substituted cognates did not inhibit the conversion, whereas an anti-P8 antibody recognizing the above sequence in PrP(C) reduced the accumulation of PrP(Sc) after 10 days of incubation of the cells. These results suggest the segment 100 to 104 of MoPrP(C) plays a key role in conversion after binding to MoPrP(Sc).     

Role of lipid rafts in the processing of the pathogenic prion and Alzheimer's amyloid-beta proteins

The conformational conversion of the cellular form of the prion protein (PrP C) into the infectious form (PrP Sc) and the proteolytic processing of the amyloid-beta (Abeta) peptide are central pathogenetic events in the prion diseases and Alzheimer's disease, respectively. Cholesterol- and sphingolipid-rich lipid rafts have emerged as important sites for the conversion of PrP C into PrP Sc, and for the proteolytic production, degradation and aggregation of Abeta. Here, we discuss these findings and their implications for our understanding of these disease processes. In addition, the potential for rafts as sites for therapeutic intervention in prion diseases and Alzheimer's disease is considered.     

Soluble dimeric prion protein binds PrP(Sc) in vivo and antagonizes prion disease

Conversion of cellular prion protein (PrP(C)) into a pathological conformer (PrP(Sc)) is thought to be promoted by PrP(Sc) in a poorly understood process. Here, we report that in wild-type mice, the expression of PrP(C) rendered soluble and dimeric by fusion to immunoglobulin Fcgamma (PrP-Fc(2)) delays PrP(Sc) accumulation, agent replication, and onset of disease following inoculation with infective prions. In infected PrP-expressing brains, PrP-Fc(2) relocates to lipid rafts and associates with PrP(Sc) without acquiring protease resistance, indicating that PrP-Fc(2) resists conversion. Accordingly, mice expressing PrP-Fc(2) but lacking endogenous PrP(C) are resistant to scrapie, do not accumulate PrP-Fc(2)(Sc), and do not transmit disease to others. These results indicate that various PrP isoforms engage in a complex in vivo, whose distortion by PrP-Fc(2) affects prion propagation and scrapie pathogenesis. The unique properties of PrP-Fc(2) suggest that soluble PrP derivatives may represent a new class of prion replication antagonists.     

Prions

The discovery of infectious proteins, denoted prions, was unexpected. After much debate over the chemical basis of heredity, resolution of this issue began with the discovery that DNA, not protein, from pneumococcus was capable of genetically transforming bacteria (Avery et al. 1944). Four decades later, the discovery that a protein could mimic viral and bacterial pathogens with respect to the transmission of some nervous system diseases (Prusiner 1982) met with great resistance. Overwhelming evidence now shows that Creutzfeldt-Jakob disease (CJD) and related disorders are caused by prions. The prion diseases are characterized by neurodegeneration and lethality. In mammals, prions reproduce by recruiting the normal, cellular isoform of the prion protein (PrP(C)) and stimulating its conversion into the disease-causing isoform (PrP(Sc)). PrP(C) and PrP(Sc) have distinct conformations: PrP(C) is rich in α-helical content and has little β-sheet structure, whereas PrP(Sc) has less α-helical content and is rich in β-sheet structure (Pan et al. 1993). The conformational conversion of PrP(C) to PrP(Sc) is the fundamental event underlying prion diseases. In this article, we provide an introduction to prions and the diseases they cause.     

The charge structure of helix 1 in the prion protein regulates conversion to pathogenic PrPSc

The prion diseases are transmissible neurodegenerative disorders linked to a pathogenic conformer (PrP(Sc)) of the normal prion protein (PrP(C)). Accumulation of PrP(Sc) occurs via a poorly defined process in which PrP(Sc) complexes with and converts endogenous PrP(C) to nascent PrP(Sc). Recent experiments have focused on the highly charged first alpha helix (H1) of PrP. It has been proposed that two putative asparagine-to-arginine intrahelical salt bridges stabilize H1 in PrP(C) yet form intermolecular ionic bonds with adjacent PrP molecules during conversion of PrP(C) to PrP(Sc) (M. P. Morrissey and E. I. Shakhnovich, Proc. Natl. Acad. Sci. USA 96:11293-11298, 1999). Subsequent work (J. O. Speare et al., J. Biol. Chem. 278:12522-12529, 2003 using a cell-free assay of PrP(Sc) conversion suggested that rather than promoting conversion, the salt bridges stabilize PrP(C) against it. However, the role of individual H1 charges in PrP(Sc) generation has not yet been investigated. To approach this question, we systematically reversed or neutralized each charged residue in H1 and tested the effect on conversion to PrP(Sc) in scrapie-infected murine neuroblastoma (ScN2a) cells. We find that replacements of charged H1 residues with like charges permit conversion, while charge reversals hinder it. Neutralization of charges in the N-terminal (amino acids 143 to 146) but not the C-terminal (amino acids 147 to 151) half of H1 permits conversion, while complete reversal of charge orientation of the putative salt bridges produces a nonconvertible PrP. Circular dichroism spectroscopy studies and confocal microscopy immunofluorescence localization studies indicated that charge substitutions did not alter the secondary structure or cell surface expression of PrP(C). These data support the necessity of specific charge orientations in H1 for a productive PrP(Sc)-PrP(C) complex.     

Proteasomal dysfunction and endoplasmic reticulum stress enhance trafficking of prion protein aggregates through the secretory pathway and increase accumulation of pathologic prion protein

A conformational change of the cellular prion protein (PrP(c)) underlies formation of PrP(Sc), which is closely associated with pathogenesis and transmission of prion diseases. The precise conformational prerequisites and the cellular environment necessary for this post-translational process remain to be completely elucidated. At steady state, glycosylated PrP(c) is found primarily at the cell surface, whereas a minor fraction of the population is disposed of by the ER-associated degradation-proteasome pathway. However, chronic ER stress conditions and proteasomal dysfunctions lead to accumulation of aggregation-prone PrP molecules in the cytosol and to neurodegeneration. In this study, we challenged different cell lines by inducing ER stress or inhibiting proteasomal activity and analyzed the subsequent repercussion on PrP metabolism, focusing on PrP in the secretory pathway. Both events led to enhanced detection of PrP aggregates and a significant increase of PrP(Sc) in persistently prion-infected cells, which could be reversed by overexpression of proteins of the cellular quality control. Remarkably, upon proteasomal impairment, an increased fraction of misfolded, fully glycosylated PrP molecules traveled through the secretory pathway and reached the plasma membrane. These findings suggest a novel pathway that possibly provides additional substrate and template necessary for prion formation when protein clearance by the proteasome is impaired.     

Toward molecular dissection of PrPC-PrPSc interactions

Direct interaction between endogenous cellular prion protein (PrP(C)) and misfolded, disease-associated (PrP(Sc)) conformers is a key event in prion propagation, which precedes templated conversion of PrP(C) into nascent PrP(Sc) and prion infectivity. Although almost none of the molecular details of this pivotal process are understood, the persistence of individual prion strains suggests that assembly of the prion replicative complex is mechanistically precise. To systematically map defined regions of PrP(C) sequence that bind tightly to PrP(Sc), we have generated a comprehensive panel of over 45 motif-grafted antibodies containing overlapping peptide grafts collectively spanning PrP residues 19-231. Grafted antibody binding experiments, performed under stringent conditions, clearly identified only three distinct and independent high affinity PrP(Sc) recognition motifs. The first of these binding motifs lies at the very N-terminal region of the mature PrP molecule within PrP-(23-33); the second motif lies within PrP-(98-110); and the third is contained within PrP-(136-158). Mutational analyses of these PrP(Sc)-binding regions revealed that reactivity of the 23-33 and 98-110 segments are largely dependent upon the presence of multiple positively charged amino acid residues. These studies yield new insight into critical peptidic components composing one side of the prion replicative interface.     

Concealment of epitope by reduction and alkylation in prion protein

Conversion of the cellular prion protein (PrP(C)) into its pathological isoform (PrP(Sc)), the key molecular event in the pathogenesis of prion diseases, is accompanied by a conformational transition of alpha-helix into beta-sheet structures involving alpha-helix 1 (alpha1) domain from residues 144 to 154 of the protein. Reduction and alkylation of PrP(C) have been found to inhibit the conversion of PrP(C) into PrP(Sc) in vitro. Here we report that while antibody affinity of epitopes in the N- and C-terminal domains remained unchanged, reduction and alkylation of the PrP molecule induced complete concealment of an epitope in alpha1 for anti-PrP antibody 6H4 that is able to cure prion infection in the cell model. Mass spectrometric analysis of recombinant PrP showed that the alkylation reaction takes place at reduced cysteines but no modification was observed in this cryptic epitope. Our study suggests that reduction and alkylation result in local or global rearrangement of PrP tertiary structure that is maintained in both liquid and solid phases. The implications in the conversion of PrP(C) into PrP(Sc) and the therapeutics of prion diseases are discussed.     

Lactoferrin induces cell surface retention of prion protein and inhibits prion accumulation

Prion diseases are fatal neurodegenerative disorders, and the conformational conversion of normal cellular prion protein (PrP(C)) into its pathogenic, amyloidogenic isoform (PrP(Sc)) is the essential event in the pathogenesis of these diseases. Lactoferrin (LF) is a cationic iron-binding glycoprotein belonging to the transferrin (TF) family, which accumulates in the amyloid deposits in the brain in neurodegenerative disorders, such as Alzheimer's disease and Pick's disease. In the present study, we have examined the effects of LF on PrP(Sc) formation by using cell culture models. Bovine LF inhibited PrP(Sc) accumulation in scrapie-infected cells in a time- and dose-dependent manner, whereas TF was not inhibitory. Bioassays of LF-treated cells demonstrated prolonged incubation periods compared with non-treated cells indicating a reduction of prion infectivity. LF mediated the cell surface retention of PrP(C) by diminishing its internalization and was capable of interacting with PrP(C) in addition to PrP(Sc). Furthermore, LF partially inhibited the formation of protease-resistant PrP as determined by the protein misfolding cyclic amplification assay. Our results suggest that LF has multifunctional antiprion activities.