The CNS glycoprotein Shadoo has PrP(C)-like protective properties and displays reduced levels in prion infections

The cellular prion protein, PrP(C), is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS-expressed Doppel or internally deleted PrP ('DeltaPrP'). This paradigm has facilitated mapping of activity determinants in PrP(C) and implicated a cryptic PrP(C)-like protein, 'pi'. Shadoo (Sho) is a hypothetical GPI-anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N-terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrP(C), but is low in cerebellar granular neurons (CGNs) containing PrP(C) and high in PrP(C)-deficient dendritic processes. In Prnp(0/0) CGNs, Sho transgenes were PrP(C)-like in their ability to counteract neurotoxic effects of either Doppel or DeltaPrP. Additionally, prion-infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for pi, and since it engenders a PrP(C)-like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology.     

Mammalian prion proteins: enigma,variation and vaccination

Although misfolding of the cellular prion protein PrP(C) into an alternative form, denoted PrP(Sc), is a key event in prion infections, the normal function of PrP(C) remains to be clearly defined. Many PrP(C)-binding proteins have been identified, but authentication of these interactions in functional assays is incomplete. Doppel (Dpl), a recently discovered PrP-like protein, might provide a new avenue by which to explore physiological and pathological functions of PrP. For example, overexpression of Dpl causes apoptotic cerebellar cell death that is abrogated by PrP(C), indicating that these two proteins can act in a common pathway. Despite our incomplete understanding of PrP(C), immunological targeting of this PrP(Sc) precursor has produced encouraging results, indicating a potential point of intervention against these fatal diseases.     

Expression of the Prion Protein Family Member Shadoo Causes Drug Hypersensitivity That Is Diminished by the Coexpression of the Wild Type Prion Protein

The prion protein (PrP) seems to exert both neuroprotective and neurotoxic activities. The toxic activities are associated with the C-terminal globular parts in the absence of the flexible N terminus, specifically the hydrophobic domain (HD) or the central region (CR). The wild type prion protein (PrP-WT), having an intact flexible part, exhibits neuroprotective qualities by virtue of diminishing many of the cytotoxic effects of these mutant prion proteins (PrPΔHD and PrPΔCR) when coexpressed. The prion protein family member Doppel, which possesses a three-dimensional fold similar to the C-terminal part of PrP, is also harmful to neuronal and other cells in various models, a phenotype that can also be eliminated by the coexpression of PrP-WT. In contrast, another prion protein family member, Shadoo (Sho), a natively disordered protein possessing structural features similar to the flexible N-terminal tail of PrP, exhibits PrP-WT-like protective properties. Here, we report that, contrary to expectations, Sho expression in SH-SY5Y or HEK293 cells induces the same toxic phenotype of drug hypersensitivity as PrPΔCR. This effect is exhibited in a dose-dependent manner and is also counteracted by the coexpression of PrP-WT. The opposing effects of Shadoo in different model systems revealed here may be explored to help discern the relationship of the various toxic activities of mutant PrPs with each other and the neurotoxic effects seen in neurodegenerative diseases, such as transmissible spongiform encephalopathy and Alzheimer disease.     

Down-regulation of Shadoo in prion infections traces a pre-clinical event inversely related to PrP(Sc) accumulation

During prion infections of the central nervous system (CNS) the cellular prion protein, PrP(C), is templated to a conformationally distinct form, PrP(Sc). Recent studies have demonstrated that the Sprn gene encodes a GPI-linked glycoprotein Shadoo (Sho), which localizes to a similar membrane environment as PrP(C) and is reduced in the brains of rodents with terminal prion disease. Here, analyses of prion-infected mice revealed that down-regulation of Sho protein was not related to Sprn mRNA abundance at any stage in prion infection. Down-regulation was robust upon propagation of a variety of prion strains in Prnp(a) and Prnp(b) mice, with the exception of the mouse-adapted BSE strain 301 V. In addition, Sho encoded by a TgSprn transgene was down-regulated to the same extent as endogenous Sho. Reduced Sho levels were not seen in a tauopathy, in chemically induced spongiform degeneration or in transgenic mice expressing the extracellular ADan amyloid peptide of familial Danish dementia. Insofar as prion-infected Prnp hemizygous mice exhibited accumulation of PrP(Sc) and down-regulation of Sho hundreds of days prior to onset of neurologic symptoms, Sho depletion can be excluded as an important trigger for clinical disease or as a simple consequence of neuronal damage. These studies instead define a disease-specific effect, and we hypothesize that membrane-associated Sho comprises a bystander substrate for processes degrading PrP(Sc). Thus, while protease-resistant PrP detected by in vitro digestion allows post mortem diagnosis, decreased levels of endogenous Sho may trace an early response to PrP(Sc) accumulation that operates in the CNS in vivo. This cellular response may offer new insights into the homeostatic mechanisms involved in detection and clearance of the misfolded proteins that drive prion disease pathogenesis.     

Protease-resistant prions selectively decrease Shadoo protein

The central event in prion diseases is the conformational conversion of the cellular prion protein (PrP(C)) into PrP(Sc), a partially protease-resistant and infectious conformer. However, the mechanism by which PrP(Sc) causes neuronal dysfunction remains poorly understood. Levels of Shadoo (Sho), a protein that resembles the flexibly disordered N-terminal domain of PrP(C), were found to be reduced in the brains of mice infected with the RML strain of prions [1], implying that Sho levels may reflect the presence of PrP(Sc) in the brain. To test this hypothesis, we examined levels of Sho during prion infection using a variety of experimental systems. Sho protein levels were decreased in the brains of mice, hamsters, voles, and sheep infected with different natural and experimental prion strains. Furthermore, Sho levels were decreased in the brains of prion-infected, transgenic mice overexpressing Sho and in infected neuroblastoma cells. Time-course experiments revealed that Sho levels were inversely proportional to levels of protease-resistant PrP(Sc). Membrane anchoring and the N-terminal domain of PrP both influenced the inverse relationship between Sho and PrP(Sc). Although increased Sho levels had no discernible effect on prion replication in mice, we conclude that Sho is the first non-PrP marker specific for prion disease. Additional studies using this paradigm may provide insight into the cellular pathways and systems subverted by PrP(Sc) during prion disease.     

Did the prion protein become vulnerable to misfolding after an evolutionary divide and conquer event?

Despite high sequence identity among mammalian prion proteins (PrPs), mammals have varying rates of susceptibility to prion disease resulting in a so-called species barrier. The species barrier follows no clear pattern, with closely related species or similar sequences being no more likely to infect each other, and remains an unresolved enigma. Variation of the conformationally flexible regions may alter the thermodynamics of the conformational change, commonly referred to as the conformational conversion, which occurs in the pathogenic process of the mammalian prion protein. A conformational ensemble scenario is supported by the species barrier in prion disease and evidence that there are strains of pathogenic prion with different conformations within species. To study how conformational flexibility has evolved in the prion protein, an investigation was undertaken on the evolutionary dynamics of structurally disordered regions in the mammalian prion protein, non-mammalian prion protein that is not vulnerable to prion disease, and remote homologs Doppel and Shadoo. Structural disorder prediction analyzed in an evolutionary context revealed that the occurrence of increased or altered conformational flexibility in mammalian PrPs coincides with key events among PrP, Doppel, and Shadoo. Comparatively rapid evolutionary dynamics of conformational flexibility in the prion protein suggest that the species barrier is not a static phenomenon. A small number of amino acid substitutions can repopulate the conformational ensemble and have a disproportionately large effect on pathogenesis.     

Is prnt a Pseudogene? Identification of Ram Prt in Testis and Ejaculated Spermatozoa

A hallmark of prion diseases or transmissible spongiform encephalopaties is the conversion of the cellular prion protein (PrP(C)), expressed by the prion gene (prnp), into an abnormally folded isoform (PrP(Sc)) with amyloid-like features that causes scrapie in sheep among other diseases. prnp together with prnd (which encodes a prion-like protein designated as Doppel), and prnt (that encodes the prion protein testis specific - Prt) with sprn (shadow of prion protein gene, that encodes Shadoo or Sho) genes, constitute the "prion gene complex". Whereas a role for prnd in the proper functioning of male reproductive system has been confirmed, the function of prnt, a recently discovered prion family gene, comprises a conundrum leading to the assumption that ruminant prnt is a pseudogene with no protein expression. The main objective of the present study was to identify Prt localization in the ram reproductive system and simultaneously to elucidate if ovine prnt gene is transcribed into protein-coding RNA. Moreover, as Prt is a prnp-related protein, the amyloid propensity was also tested for ovine and caprine Prt. Recombinant Prt was used to immunize BALB/c mice, and the anti-Prt polyclonal antibody (APPA) immune response was evaluated by ELISA and Western Blot. When tested by indirect immunofluorescence, APPA showed high avidity to the ram sperm head apical ridge subdomain, before and after induced capacitation, but did not show the same behavior against goat spermatozoa, suggesting high antibody specificity against ovine-Prt. Prt was also found in the testis when assayed by immunohistochemistry during ram spermatogenesis, where spermatogonia, spermatocytes, spermatids and spermatozoa, stained positive. These observations strongly suggest ovine prnt to be a translated protein-coding gene, pointing to a role for Prt protein in the ram reproductive physiology. Besides, caprine Prt appears to exhibit a higher amyloid propensity than ovine Prt, mostly associated with its phenylalanine residue.     

Targeting cellular prion protein reverses early cognitive deficits and neurophysiological dysfunction in prion-infected mice

Currently, no treatment can prevent the cognitive and motor decline associated with widespread neurodegeneration in prion disease. However, we previously showed that targeting endogenous neuronal prion protein (PrP(C)) (the precursor of its disease-associated isoform, PrP(Sc)) in mice with early prion infection reversed spongiform change and prevented clinical symptoms and neuronal loss. We now show that cognitive and behavioral deficits and impaired neurophysiological function accompany early hippocampal spongiform pathology. Remarkably, these behavioral and synaptic impairments recover when neuronal PrP(C) is depleted, in parallel with reversal of spongiosis. Thus, early functional impairments precede neuronal loss in prion disease and can be rescued. Further, they occur before extensive PrP(Sc) deposits accumulate and recover rapidly after PrP(C) depletion, supporting the concept that they are caused by a transient neurotoxic species, distinct from aggregated PrP(Sc). These data suggest that early intervention in human prion disease may lead to recovery of cognitive and behavioral symptoms.     

Prion proteins and infertility: insight from mouse models

A wealth of evidence points to an abnormal form of the prion protein called PrP(Sc) as the transmissible agent responsible for prion diseases. However, the physiological function of its normal conformer, the cellular prion protein (PrP(C)), is still unknown. Recently, a homologue of PrP(C) was discovered and denoted Doppel (Dpl). In contrast to PrP, mice deficient for Dpl suffer from an important pathological phenotype: male sterility. This phenotype shifts the attention from the brain, where most of the investigations on Dpl have been performed, to testis, raising hope to resolve the long lasting search of PrP(C) function.     

Purkinje-cell degeneration in prion protein-deficient mice is associated with a cerebellum-specific Doppel protein species signature

PrP(c) (cellular prion protein) and Doppel are antagonizing proteins, respectively neuroprotective and neurotoxic. Evidence for Doppel neurotoxicity came from PrP(c)-deficient (Prnp(0/0)) mouse lines developing late onset Purkinje-cell degeneration caused by Doppel overexpression in brain. To address the molecular underpinnings of this cell-type specificity, we generated Doppel N-terminal-specific antibodies and started to examine the spatio-temporal expression of Doppel protein species in Ngsk Prnp(0/0) brain. Although Doppel overexpression is ubiquitous, Western analyses of normal and deglycosylated protein extracts revealed cerebellar patterns distinct from the rest of the brain, supporting the idea that neurotoxicity might be linked to a particular Doppel species pattern. Furthermore, our newly raised antibodies allowed the first Doppel immunohistochemical analyses in brain, showing a distribution in Prnp(0/0) cerebellum similar to PrP(c) in wild type.     

Differences between the prion protein and its homolog Doppel: a partially structured state with implications for scrapie formation

The key event in the pathogenesis of prion diseases is a conformational change in the prion protein (PrP). Models for conversion of PrP(C) into PrP(Sc) typically implicate an, as yet, unidentified intermediate. In an attempt to identify such an intermediate, we used native-state hydrogen exchange monitored with NMR. Although we were unable to detect an intermediate directly, we observed substantial protection above that expected based upon measurements of the global stability of PrP (>2 kcal mol(-1) super protection). This super protection implicates either structure in the denatured state or the presence of an intermediate. Similar experiments with Doppel, a homolog of PrP that does not form infectious prions, failed to demonstrate such super protection. This suggests that the partially structured state of PrP encompassing portions of the B and C helices, may be a significant factor in the ability of PrP to convert from PrP(C) to 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.     

The prion gene complex encoding PrP(C) and Doppel: insights from mutational analysis

The prion protein gene, Prnp, encodes PrP(Sc), the major structural component of prions, infectious pathogens causing a number of disorders including scrapie and bovine spongiform encephalopathy (or BSE). Missense mutations in the human Prnp gene cause inherited prion diseases such as familial Creutzfeldt-Jakob disease. In uninfected animals Prnp encodes a glycophosphatidylinositol (GPI)-anchored protein denoted PrP(C) and in prion infections PrP(C) is converted to PrP(Sc) by templated refolding. Though Prnp is conserved in mammalian species, attempts to verify interactions of putative PrP binding proteins by genetic means have proven frustrating and the ZrchI and Npu lines of Prnp gene-ablated mice (Prnp(0/0) mice) lacking PrP(C) remain healthy throughout development. This indicates that PrP(C) serves a function that is not apparent in a laboratory setting or that other molecules have overlapping functions. Current possibilities involve shuttling or sequestration of synaptic Cu(II) via binding to N-terminal octapeptide residues and/or signal transduction involving the fyn kinase. A new point of entry into the issue of prion protein function has emerged from identification of a paralogue, Prnd, with 24% coding sequence identity to Prnp. Prnd lies downstream of Prnp and encodes the doppel (Dpl) protein. Like PrP(C), Dpl is presented on the cell surface via a GPI anchor and has three alpha-helices: however, it lacks the conformationally plastic and octapeptide repeat domains present in its well-known relative. Interestingly, Dpl is overexpressed in the Ngsk and Rcm0 lines of Prnp(0/0) mice via intergenic splicing events. These lines of Prnp(0/0) mice exhibit ataxia and apoptosis of cerebellar cells, indicating that ectopic synthesis of Dpl protein is toxic to central nervous system neurons: this inference has now been confirmed by the construction of transgenic mice expressing Dpl under the direct control of the PrP promoter. Remarkably, Dpl-programmed ataxia is rescued by wild-type Prnp transgenes. The interaction between the Prnp and Prnd genes in mouse cerebellar neurons may have a physical correlate in competition between Dpl and PrP(C) within a common biochemical pathway that when mis-regulated leads to apoptosis.     

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.     

Novel differences between two human prion strains revealed by two-dimensional gel electrophoresis

The phenotype of human sporadic prion diseases is affected by patient genotype at codon 129 of the prion protein (PrP) gene, the site of a common methionine/valine polymorphism, and by the type of the scrapie PrP (PrP(Sc)), which likely reflects the prion strain. However, two distinct disease phenotypes, identified as sporadic Creutzfeldt-Jakob disease (M/M2 sCJD) and sporadic fatal insomnia (sFI), share methionine homozygosity at codon 129 and PrP(Sc) type 2. One-dimensional gel electrophoresis and immunoblotting reveal no difference between the M/M2 sCJD and sFI species of PrP(Sc) in gel mobility and glycoform ratio. In contrast, the two-dimensional immunoblot demonstrates that in M/M2 sCJD the full-length PrP(Sc) form is overrepresented and carries glycans that are different from those present in the PrP(Sc) of sFI. Because the altered glycans are detectable only in the PrP(Sc) and not in the normal or cellular PrP (PrP(C)), they are likely to result from preferential conversion to PrP(Sc) of rare PrP(C) glycoforms. This is the first evidence that a qualitative difference in glycans contributes to prion diversity.     

Cell-free propagation of prion strains

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). Disease is transmitted by the autocatalytic propagation of PrP(Sc) misfolding at the expense of the normal prion protein. The biggest challenge of the prion hypothesis has been to explain the molecular mechanism by which prions can exist as different strains, producing diseases with distinguishable characteristics. Here, we show that PrP(Sc) generated in vitro by protein misfolding cyclic amplification from five different mouse prion strains maintains the strain-specific properties. Inoculation of wild-type mice with in vitro-generated PrP(Sc) caused a disease with indistinguishable incubation times as well as neuropathological and biochemical characteristics as the parental strains. Biochemical features were also maintained upon replication of four human prion strains. These results provide additional support for the prion hypothesis and indicate that strain characteristics can be faithfully propagated in the absence of living cells, suggesting that strain variation is dependent on PrP(Sc) properties.     

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.     

Seeded conversion of recombinant prion protein to a disulfide-bonded oligomer by a reduction-oxidation process

The infectious form of prion protein, PrP(Sc), self-propagates by its conversion of the normal, cellular prion protein molecule PrP(C) to another PrP(Sc) molecule. It has not yet been demonstrated that recombinant prion protein can convert prion protein molecules from PrP(C) to PrP(Sc). Here we show that recombinant hamster prion protein is converted to a second form, PrP(RDX), by a redox process in vitro and that this PrP(RDX) form seeds the conversion of other PrP(C) molecules to the PrP(RDX) form. The converted form shows properties of oligomerization and seeded conversion that are characteristic of PrP(Sc). We also find that the oligomerization can be reversed in vitro. X-ray fiber diffraction suggests an amyloid-like structure for the oligomerized prion protein. A domain-swapping model involving intermolecular disulfide bonds can account for the stability and coexistence of two molecular forms of prion protein and the capacity of the second form for self-propagation.     

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).