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).     

Disease-related prion protein forms aggresomes in neuronal cells leading to caspase activation and apoptosis

The molecular basis for neuronal death in prion disease is not established, but putative pathogenic roles for both disease-related prion protein (PrP(Sc)) and accumulated cytosolic PrP(C) have been proposed. Here we report that only prion-infected neuronal cells become apoptotic after mild inhibition of the proteasome, and this is strictly dependent upon sustained propagation of PrP(Sc). Whereas cells overexpressing PrP(C) developed cytosolic PrP(C) aggregates, this did not cause cell death. In contrast, only in prion-infected cells, mild proteasome impairment resulted in the formation of large cytosolic perinuclear aggresomes that contained PrP(Sc), heat shock chaperone 70, ubiquitin, proteasome subunits, and vimentin. Similar structures were found in the brains of prion-infected mice. PrP(Sc) aggresome formation was directly associated with activation of caspase 3 and 8, resulting in apoptosis. These data suggest that neuronal propagation of prions invokes a neurotoxic mechanism involving intracellular formation of PrP(Sc) aggresomes. This, in turn, triggers caspase-dependent apoptosis and further implicates proteasome dysfunction in the pathogenesis of prion diseases.     

Ablation of the metal ion-induced endocytosis of the prion protein by disease-associated mutation of the octarepeat region.

The neurodegenerative spongiform encephalopathies, or prion diseases, are characterized by the conversion of the normal cellular form of the prion protein PrP(C) to a pathogenic form, PrP(Sc) [1]. There are four copies of an octarepeat PHGG(G/S)WGQ that specifically bind Cu(2+) ions within the N-terminal half of PrP(C) [2--4]. This has led to proposals that prion diseases may, in part, be due to abrogation of the normal cellular role of PrP(C) in copper homeostasis [5]. Here, we show that murine PrP(C) is rapidly endocytosed upon exposure of neuronal cells to physiologically relevant concentrations of Cu(2+) or Zn(2+), but not Mn(2+). Deletion of the four octarepeats or mutation of the histidine residues (H68/76 dyad) in the central two repeats abolished endocytosis, indicating that the internalization of PrP(C) is governed by metal binding to the octarepeats. Furthermore, a mutant form of PrP that contains nine additional octarepeats and is associated with familial prion disease [6] failed to undergo Cu(2+)-mediated endocytosis. For the first time, these results provide evidence that metal ions can promote the endocytosis of a mammalian prion protein in neuronal cells and that neurodegeneration associated with some prion diseases may arise from the ablation of this function due to mutation of the octarepeat region.     

Prion protein with an octapeptide insertion has impaired neuroprotective activity in transgenic mice

Familial prion diseases are due to dominantly inherited, germline mutations in the PRNP gene that encodes the prion protein (PrP). The cellular mechanism underlying the pathogenic effect of these mutations remains uncertain. To investigate whether pathogenic mutations impair a normal, physiological activity of PrP, we have crossed Tg(PG14) mice, which express PrP with an octapeptide insertion associated with an inherited prion dementia, with Tg(PrPDelta32-134) mice. Tg(PrPDelta32-134) mice, which express an N-terminally truncated form of PrP, spontaneously develop a neurodegenerative phenotype that is stoichiometrically reversed by coexpression of wild-type PrP. We find that, at equivalent expression levels, PG14 PrP is significantly less efficient than wild-type PrP in suppressing the development of clinical symptoms and neuropathology in Tg(PrPDelta32-134) mice. Thus, our results suggest that some features of the neurological illness associated with inherited PrP mutations may be attributable to a loss of PrP neuroprotective function. This mechanism stands in contrast to the toxic gain-of-function mechanisms that are usually invoked to explain the pathogenesis of dominantly inherited neurodegenerative disorders.     

In vitro self-propagation of recombinant PrPSc-like conformation generated in the yeast cytoplasm

Self-propagation is characteristic property for a prion conformation. Previous studies revealed that prion protein expressed in the cytoplasm gained a PrP(Sc)-like conformation. However, it remains unclear whether the PrP(Sc)-like conformation has the self-propagating property. We found that PrP partially purified from yeast cytoplasm formed amyloid fiber like structures, and we found that the PrP(Sc)-like conformation is able to convert normal PrP(C) in the brain homogenate to a proteinase K-resistant conformation. These results suggest that yeast cytoplasm expressed recombinant PrP(Sc)-like conformation has the characteristic self-propagating property of a prion, which may have implications in the pathogenesis of sporadic and inherited prion diseases.     

Prion protein aggresomes are poly(A)+ ribonucleoprotein complexes that induce a PKR-mediated deficient cell stress response

In mammalian cells, cytoplasmic protein aggregates generally coalesce to form aggresomal particles. Recent studies indicate that prion-infected cells produce prion protein (PrP) aggresomes, and that such aggregates may be present in the brain of infected mice. The molecular activity of PrP aggresomes has not been fully investigated. We report that PrP aggresomes initiate a cell stress response by activating the RNA-dependent protein kinase (PKR). Activated PKR phosphorylates the translation initiation factor eIF2alpha, resulting in protein synthesis shut-off. However, other components of the stress response, including the assembly of poly(A)+ RNA-containing stress granules and the synthesis of heat shock protein 70, are repressed. In situ hybridization experiments and affinity chromatography on oligo(dT)-cellulose showed that PrP aggresomes bind poly(A)+ RNA, and are therefore poly(A)+ ribonucleoprotein complexes. These findings support a model in which PrP aggresomes send neuronal cells into untimely demise by modifying the cell stress response, and by inducing the aggregation of poly(A)+ RNA.     

Prion protein with an E200K mutation displays properties similar to those of the cellular isoform PrP(C)

Creutzfeldt-Jakob disease (CJD) in Libyan Jews, linked to the E200K mutation in PRNP (E200KCJD), is the most prevalent of the inherited prion diseases. As other prion diseases, E200KCJD is characterized by the brain accumulation of PrP(Sc), a pathologic conformational isoform of a normal glycoprotein denominated PrP(C). To investigate whether the E200K mutation is enough to de novo confer PrP(Sc) properties to mutant PrP, as suggested by experiments in Chinese hamster ovary cells, we examined the biochemical behavior of E200KPrP in brains and fibroblasts from sporadic as well as homozygous and heterozygous E200KCJD patients, asymptomatic transgenic mice carrying the E200K mutation, as well as in normal and scrapie-infected mouse neuroblastoma cells expressing E200KPrP. E200KPrP was examined for protease sensitivity, solubility in detergents, releasibility by phosphoinositol phospholypase-C and localization in cholesterol enriched membrane microdomains (rafts). In all tissues except in brains of CJD patients and ScN2a cells, E200KPrP displayed properties similar to those of PrP(C). Our results indicate that the E200K mutation does not automatically convey the properties of PrP(Sc) to new PrP molecules. A conversion process occurs mainly in the prion disease affected brain, suggesting the presence of a tissue-specific or age-dependent factor, in accord with the late onset nature of inherited CJD.     

Production of cattle lacking prion protein

Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrP(C), such as PrP(BSE) in bovine spongiform encephalopathy (BSE) in cattle and PrP(CJD) in Creutzfeldt-Jakob disease (CJD) in humans. Disruption of PrP(C) expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities. However, the impact of ablating PrP(C) function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrP(C)-deficient cattle produced by a sequential gene-targeting system. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification. PrP(C)-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins.     

Motor behavioral and neuropathological deficits in mice deficient for normal prion protein expression

It has been difficult to reconcile the absence of pathology and apparently normal behavior of mice lacking prion protein (PrP), referred to as Prnp(0/0) mice, with a mechanism of prion pathogenesis involving progressive loss of PrP(C)-mediated neuroprotection. However, here we report that Prnp(0/0) mice exhibit significant age-related defects in motor coordination and balance compared with mice expressing wild type Prnp on a syngeneic background, and that the brains of behaviorally-impaired Prnp(0/0) mice display the cardinal neuropathological hallmarks of spongiform pathology and reactive astrocytic gliosis that normally accompany prion disease. Consistent with the appearance of cerebellar ataxia as an early symptom in patients with Gerstmann-Str?ussler-Scheinker syndrome (GSS), an inherited form of human prion disease, motor coordination and balance defects manifested in a transgenic (Tg) mouse model of GSS considerably earlier than the onset of end-stage neurodegenerative disease. Our results are consistent with a mechanism in which loss of normal PrP(C) function is an important pathological component of prion diseases.     

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.     

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.     

Prion protein repeat expansion results in increased aggregation and reveals phenotypic variability

Mammalian prion diseases are fatal neurodegenerative disorders dependent on the prion protein PrP. Expansion of the oligopeptide repeats (ORE) found in PrP is associated with inherited prion diseases. Patients with ORE frequently harbor PrP aggregates, but other factors may contribute to pathology, as they often present with unexplained phenotypic variability. We created chimeric yeast-mammalian prion proteins to examine the influence of the PrP ORE on prion properties in yeast. Remarkably, all chimeric proteins maintained prion characteristics. The largest repeat expansion chimera displayed a higher propensity to maintain a self-propagating aggregated state. Strikingly, the repeat expansion conferred increased conformational flexibility, as observed by enhanced phenotypic variation. Furthermore, the repeat expansion chimera displayed an increased rate of prion conversion, but only in the presence of another aggregate, the [RNQ+] prion. We suggest that the PrP ORE increases the conformational flexibility of the prion protein, thereby enhancing the formation of multiple distinct aggregate structures and allowing more frequent prion conversion. Both of these characteristics may contribute to the phenotypic variability associated with PrP repeat expansion diseases.     

The cellular prion protein mediates neurotoxic signalling of β-sheet-rich conformers independent of prion replication

Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimer's disease or prion diseases. Moreover, increasing evidence indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease-associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrP(Sc)), a β-sheet-rich isoform of the cellular PrP (PrP(C)), are dependent on neuronal expression of PrP(C). In this study, we demonstrate that PrP(C) has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various β-sheet-rich (β) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid β-peptide, (iii) yeast prion proteins or (iv) designed β-peptides. Toxic signalling via PrP(C) requires the intrinsically disordered N-terminal domain (N-PrP) and the GPI anchor of PrP. We found that the N-terminal domain is important for mediating the interaction of PrP(C) with β-conformers. Interestingly, a secreted version of N-PrP associated with β-conformers and antagonized their toxic signalling via PrP(C). Moreover, PrP(C)-mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer-specific antibody. Our study indicates that PrP(C) can mediate toxic signalling of various β-sheet-rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases.     

The role of prion peptide structure and aggregation in toxicity and membrane binding

Prion diseases are neurodegenerative disorders associated with a conformational change in the normal cellular isoform of the prion protein, PrP(C), to an abnormal scrapie isoform, PrP(SC). Unlike the alpha-helical PrP(C), the protease-resistant core of PrP(SC) is predominantly beta-sheet and possesses a tendency to polymerize into amyloid fibrils. We performed experiments with two synthetic human prion peptides, PrP(106-126) and PrP(127-147), to determine how peptide structure affects neurotoxicity and protein-membrane interactions. Peptide solutions possessing beta-sheet and amyloid structures were neurotoxic to PC12 cells in vitro and bound with measurable affinities to cholesterol-rich phospholipid membranes at ambient conditions, but peptide solutions lacking stable beta-sheet structures and amyloid content were nontoxic and possessed less than one tenth of the binding affinities of the amyloid-containing peptides. Regardless of structure, the peptide binding affinities to cholesterol-depleted membranes were greatly reduced. These results suggest that the beta-sheet and amyloid structures of the prion peptides give rise to their toxicity and membrane binding affinities and that membrane binding affinity, especially in cholesterol-rich environments, may be related to toxicity. Our results may have significance in understanding the role of the fibrillogenic cerebral deposits associated with some of the prion diseases in neurodegeneration and may have implications for other amyloidoses.     

Prion diseases: copper deficiency states associated with impaired nitrogen monoxide or carbon monoxide transduction and translocation.

Literature concerning prion diseases and Cu metabolism was examined to determine merits of various suggestions concerning the relationship between these diseases and altered Cu metabolism. There are a number of recent suggestions that the normal non-pathogenic form of the prion protein (PrP(C)) contains Cu while the abnormal pathogenic form of this protein, PrP(SC), lacks Cu. Results of experiments showing oxidant sensitivity in the presence of ionically bonded Cu and millimolar concentrations of hydrogen peroxide were found to lack relevance. Demonstrating superoxide disproportionation and a correlation with cellular Cu2Zn2SOD activity is relevant and consistent with a role for PrP(C) in Cu endocytosis. There are also a number of recent suggestions that PrP(C) has a role in nerve transmission. Serum from mice that lack cellular PrP(C) was found to have an elevated Cu content consistent with a response to overcome an inflammatory disease. Attempts to induce a 'transmissible' form of prion disease requiring intracerebral injections of somewhat purified brain homogenates were found lacking in support for an etiology occurring as the result of oral ingestion of supposedly 'infected' tissues. It is suggested that PrP(C) is a normal Cu-dependent cuproglycoprotein of unknown function that may have a role in facilitating normal nitrogen monoxide- or carbon monoxide-mediated biochemistry.     

Novel heparan mimetics potently inhibit the scrapie prion protein and its endocytosis

During prion diseases the normal prion protein PrP(C) is refolded into an abnormal conformer PrP(Sc). We have studied the PrP(Sc) inhibiting activity of a library of synthetic heparan mimetic (HM) biopolymers. HMs are chemically derived dextrans obtained by successive substitutions with carboxymethyl, benzylamide, and sulfate groups on glucose residues. Some HMs eliminated PrP(Sc) from prion-infected cells after a 5 day course at 100 ng/ml and were 15 x potent than pentosan sulfate in this system. The anti-PrP(Sc) activity of HMs correlated with the degree of sulfation but was increased by benzylamidation. HMs did not reduce the synthesis of PrP(C) nor its attachment to lipid rafts, but instead blocked its conversion into PrP(Sc). The anti-PrP(Sc) HMs also prevented the uptake of prion rods by cultured cells. HMs may thus block the interaction of PrP(Sc) with a putative cellular receptor, possibly heparan sulfate. HMs provide an attractive chemical approach for the synthesis of TSE therapeutic and prophylactic reagents.     

Trapping prion protein in the endoplasmic reticulum impairs PrPC maturation and prevents PrPSc accumulation

The conversion of the normal cellular prion protein (PrP(C)) into the abnormal scrapie isoform (PrP(Sc)) is a key feature of prion diseases. The pathogenic mechanisms and the subcellular sites of the conversion are complex and not completely understood. In particular, little is known on the role of the early compartment of the secretory pathway in the processing of PrP(C) and in the pathogenesis of prion diseases. In order to interfere with the intracellular traffic of endogenous PrP(C) we have generated two anti-prion single chain antibody fragments (scFv) directed against different epitopes, each fragment tagged either with a secretory leader or with the ER retention signal KDEL. The stable expression of these constructs in PC12 cells allowed us to study their specific effects on the synthesis, maturation, and processing of endogenous PrP(C) and on PrP(Sc) formation. We found that ER-targeted anti-prion scFvs retain PrP(C) in the ER and inhibit its translocation to the cell surface. Retention in the ER strongly affects the maturation and glycosylation state of PrP(C), with the appearance of a new aberrant endo-H sensitive glycosylated species. Interestingly, ER-trapped PrP(C) acquires detergent insolubility and proteinase K resistance. Furthermore, we show that ER-targeted anti-prion antibodies prevent PrP(Sc) accumulation in nerve growth factor-differentiated PC12 cells, providing a new tool to study the molecular pathology of prion diseases.     

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.     

Systematic identification of antiprion drugs by high-throughput screening based on scanning for intensely fluorescent targets

Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrP(Sc)), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrP(C)/PrP(Sc) interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 microM. Among these, six compounds displayed an inhibitory effect on PrP(Sc) propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N'-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrP(C) and PrP(Sc) is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.     

Proteasomes and ubiquitin are involved in the turnover of the wild-type prion protein

Prion diseases propagate by converting a normal glycoprotein of the host, PrP(C), into a pathogenic "prion" conformation. Several misfolding mutants of PrP(C) are degraded through the ER-associated degradation (ERAD)-proteasome pathway. In their infectious form, prion diseases such as bovine spongiform encephalopathy involve PrP(C) of wild-type sequence. In contrast to mutant PrP, wild-type PrP(C) was hitherto thought to be stable in the ER and thus immune to ERAD. Using proteasome inhibitors, we now show that approximately 10% of nascent PrP(C) molecules are diverted into the ERAD pathway. Cells incubated with N-acetyl-leucinal-leucinal-norleucinal (ALLN), lactacystin or MG132 accumulated both detergent-soluble and insoluble PrP species. The insoluble fraction included an unglycosylated 26 kDa PrP species with a protease-resistant core, and a M(r) "ladder" that contained ubiquitylated PrP. Our results show for the first time that wild-type PrP(C) molecules are subjected to ERAD, in the course of which they are dislocated into the cytosol and ubiquitylated. The presence of wild-type PrP molecules in the cytosol may have potential pathogenic implications.