The prion protein family: diversity, rivalry, and dysfunction

The prion gene family currently consists of three members: Prnp which encodes PrP(C), the precursor to prion disease associated isoforms such as PrP(Sc); Prnd which encodes Doppel, a testis-specific protein involved in the male reproductive system; and Sprn which encodes the newest PrP-like protein, Shadoo, which is expressed in the CNS. Although the identification of numerous candidate binding partners for PrP(C) has hinted at possible cellular roles, molecular interpretations of PrP(C) activity remain obscure and no widely-accepted view as to PrP(C) function has emerged. Nonetheless, studies into the functional interrelationships of prion proteins have revealed an interesting phenomenon: Doppel is neurotoxic to cerebellar cells in a manner which can be blocked by either PrP(C) or Shadoo. Further examination of this paradigm may help to shed light on two prominent unanswered questions in prion biology: the functional role of PrP(C) and the neurotoxic pathways initiated by PrP(Sc) in prion 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.     

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.     

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.     

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.     

Genetic mapping of activity determinants within cellular prion proteins: N-terminal modules in PrPC offset pro-apoptotic activity of the Doppel helix B/B' region

The PrP-like Doppel (Dpl) protein causes apoptotic death of cerebellar neurons in transgenic mice, a process prevented by expression of the wild type (wt) cellular prion protein, PrP(C). Internally deleted forms of PrP(C) resembling Dpl such as PrPDelta32-121 produce a similar PrP(C)-sensitive pro-apoptotic phenotype in transgenic mice. Here we demonstrate that these phenotypic attributes of wt Dpl, wt PrP(C), and PrPDelta132-121 can be accurately recapitulated by transfected mouse cerebellar granule cell cultures. This system was then explored by mutagenesis of the co-expressed prion proteins to reveal functional determinants. By this means, neuroprotective activity of wt PrP(C) was shown to be nullified by a deletion of the N-terminal charged region implicated in endocytosis and retrograde axonal transport (PrPDelta23-28), by deletion of all five octarepeats (PrPDelta51-90), or by glycine replacement of four octarepeat histidine residues required for selective binding of copper ions (Prnp"H/G"). In the case of Dpl, overlapping deletions defined a requirement for the gene interval encoding helices B and B' (DplDelta101-125). These data suggest contributions of copper binding and neuronal trafficking to wt PrP(C) function in vivo and place constraints upon current hypotheses to explain Dpl/PrP(C) antagonism by competitive ligand binding. Further implementation of this assay should provide a fuller understanding of the attributes and subcellular localizations required for activity of these enigmatic proteins.     

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.     

Conserved stress-protective activity between prion protein and Shadoo

Shadoo (Sho) is a neuronally expressed glycoprotein of unknown function. Although there is no overall sequence homology to the cellular prion protein (PrP(C)), both proteins contain a highly conserved internal hydrophobic domain (HD) and are tethered to the outer leaflet of the plasma membrane via a C-terminal glycosylphosphatidylinositol anchor. A previous study revealed that Sho can reduce toxicity of a PrP mutant devoid of the HD (PrPΔHD). We have now studied the stress-protective activity of Sho in detail and identified domains involved in this activity. Like PrP(C), Sho protects cells against physiological stressors such as the excitotoxin glutamate. Moreover, both PrP(C) and Sho required the N-terminal domain for this activity; the stress-protective capacity of PrPΔN as well as ShoΔN was significantly impaired. In both proteins, the HD promoted homodimer formation; however, deletion of the HD had different effects. Although ShoΔHD lost its stress-protective activity, PrPΔHD acquired a neurotoxic potential. Finally, we could show that the N-terminal domain of PrP(C) could be functionally replaced by that of Sho, suggesting a similar function of the N termini of Sho and PrP(C). Our study reveals a conserved physiological activity between PrP(C) and Sho to protect cells from stress-induced toxicity and suggests that Sho and PrP(C) might act on similar signaling pathways.     

Human Doppel and prion protein share common membrane microdomains and internalization pathways

Doppel is the first identified homologue of the prion protein (PrPc) implicated in prion disease. Doppel is considered an N-truncated form of PrPc, and shares with PrPc several structural and biochemical features. When over expressed in the brain of some PrP knockout animals, it provokes cerebellar ataxia. As this phenotype is rescued by reintroducing the PrP gene, it has been suggested that Doppel and PrPc have antagonistic functions and may compete for a common ligand. However, a direct interaction between the two proteins has recently been observed. To investigate whether the neuronal environment is suitable for such possibility, human Doppel and PrPc were expressed separately, or together, in neuroblastoma cells, and then studied by biochemical and immunomicroscopic tools, as well as in intact cells expressing fluorescent fusion constructs. The results demonstrate that Doppel and PrPc co-patch extensively at the plasma membrane, and get internalized together after ganglioside cross-linking by cholera toxin or addition of an antibody against only one of the proteins. These processes no longer occur if the integrity of rafts is disrupted. We also show that, whereas each protein expressed alone occupies Triton X-100-insoluble membrane microdomains, co-transfected Doppel and PrPc redistribute together into a less ordered lipidic environment. All these features are consistent with interactions occurring between Doppel and PrPc in our neuronal cell model.     

Shadoo binds lipid membranes and undergoes aggregation and fibrillization

Lipid membrane can enhance prion protein (PrP) pathological fibrillogenesis. A neuronal paralog of PrP, named Shadoo (Sho), is localized to similar membrane environment as PrP and can also convert to amyloid-like fibrilles. To gain insight into the role of Sho in prion diseases, we studied Sho interactions with cellular membrane models. Sho was found to bind anionic lipid vesicles. Spectroscopic and microscopic data showed that membrane-associated Sho slowly converted into amyloid fibers. Furthermore, binding of Sho to anionic liposomes has a disruptive effect on the integrity of the lipid bilayer leading to the formation of supramolecular lipid–protein complexes. In consequence, the role of Sho in prion diseases might depend on the oligomerization state of Sho but also the nature of these lipoprotein assembles.     

The N-terminal, polybasic region is critical for prion protein neuroprotective activity

Several lines of evidence suggest that the normal form of the prion protein, PrP(C), exerts a neuroprotective activity against cellular stress or toxicity. One of the clearest examples of such activity is the ability of wild-type PrP(C) to suppress the spontaneous neurodegenerative phenotype of transgenic mice expressing a deleted form of PrP (Δ32-134, called F35). To define domains of PrP involved in its neuroprotective activity, we have analyzed the ability of several deletion mutants of PrP (Δ23-31, Δ23-111, and Δ23-134) to rescue the phenotype of Tg(F35) mice. Surprisingly, all of these mutants displayed greatly diminished rescue activity, although Δ23-31 PrP partially suppressed neuronal loss when expressed at very high levels. Our results pinpoint the N-terminal, polybasic domain as a critical determinant of PrP(C) neuroprotective activity, and suggest that identification of molecules interacting with this region will provide important clues regarding the normal function of the protein. Small molecule ligands targeting this region may also represent useful therapeutic agents for treatment of prion diseases.     

Serum withdrawal-induced apoptosis in ZrchI prion protein (PrP) gene-deficient neuronal cell line is suppressed by PrP, independent of Doppel

Previous studies have shown that cellular prion protein (PrP(C)) plays anti-apoptotic and antioxidative role against cell death induced by serum-deprivation (SDP) in an immortalized prion protein gene-deficient neuronal cell line derived from Rikn prion protein (PrP) gene-deficient (Prnp(-/-)) mice, which ectopically produce excess Doppel (Dpl) (PrP-like glycoprotein). To investigate whether PrP(C) inhibits apoptotic neuronal cell death without Dpl, an immortalized cell line was established from the brain of ZrchI Prnp(-/-) mice, which do not show ectopic expression of Dpl. The results using a ZrchI neuronal Prnp(-/-) cell line (NpL2) showed that PrP(C) potently inhibited SDP-induced apoptotic cell death. Furthermore, PrP(C) expression enhanced the superoxide dismutase (SOD) activity in NpL2 cells. These results indicate that Dpl production did not affect anti-apoptotic and anti-oxidative functions of PrP, suggesting that PrP(C) may be directly correlated with protection against oxidative stress.     

Mutant PrP suppresses glutamatergic neurotransmission in cerebellar granule neurons by impairing membrane delivery of VGCC α(2)δ-1 Subunit

How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α(2)δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α(2)δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies.     

Neonatal lethality in transgenic mice expressing prion protein with a deletion of residues 105-125

To identify sequence domains important for the neurotoxic and neuroprotective activities of the prion protein (PrP), we have engineered transgenic mice that express a form of murine PrP deleted for a conserved block of 21 amino acids (residues 105-125) in the unstructured, N-terminal tail of the protein. These mice spontaneously developed a severe neurodegenerative illness that was lethal within 1 week of birth in the absence of endogenous PrP. This phenotype was reversed in a dose-dependent fashion by coexpression of wild-type PrP, with five-fold overexpression delaying death beyond 1 year. The phenotype of Tg(PrPDelta105-125) mice is reminiscent of, but much more severe than, those described in mice that express PrP harboring larger deletions of the N-terminus, and in mice that ectopically express Doppel, a PrP paralog, in the CNS. The dramatically increased toxicity of PrPDelta105-125 is most consistent with a model in which this protein has greatly enhanced affinity for a hypothetical receptor that serves to transduce the toxic signal. We speculate that altered binding interactions involving the 105-125 region of PrP may also play a role in generating neurotoxic signals during prion infection.     

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.     

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.     

Shadoo (Sprn) and prion disease incubation time in mice

Prion diseases are transmissible neurodegenerative disorders of mammalian species and include scrapie, bovine spongiform encephalopathy (BSE), and variant Creutzfeldt-Jakob disease (vCJD). The prion protein (PrP) plays a key role in the disease, with coding polymorphism in both human and mouse influencing disease susceptibility and incubation time, respectively. Other genes are also thought to be important and a plausible candidate is Sprn, which encodes the PrP-like protein Shadoo (Sho). Sho is expressed in the adult central nervous system and exhibits neuroprotective activity reminiscent of PrP in an in vitro assay. To investigate the role of Sprn in prion disease incubation time we sequenced the open reading frame (ORF) in a diverse panel of mice and saw little variation except in strains derived from wild-trapped mice. Sequencing the untranslated regions revealed polymorphisms that allowed us to carry out an association study of incubation period in the Northport heterogeneous stock of mice inoculated with Chandler/RML prions. We also examined the expression level of Sprn mRNA in the brains of normal and prion-infected mice and saw no correlation with either genotype or incubation time. We therefore conclude that Sprn does not play a major role in prion disease incubation time in these strains of mice.     

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.     

Comparative computational analysis of prion proteins reveals two fragments with unusual structural properties and a pattern of increase in hydrophobicity associated with disease-promoting mutations

Prion diseases are a group of neurodegenerative disorders associated with conversion of a normal prion protein, PrPC, into a pathogenic conformation, PrPSc. The PrPSc is thought to promote the conversion of PrPC. The structure and stability of PrPC are well characterized, whereas little is known about the structure of PrPSc, what parts of PrPC undergo conformational transition, or how mutations facilitate this transition. We use a computational knowledge-based approach to analyze the intrinsic structural propensities of the C-terminal domain of PrP and gain insights into possible mechanisms of structural conversion. We compare the properties of PrP sequences to those of a PrP paralog, Doppel, and to the distributions of structural propensities observed in known protein structures from the Protein Data Bank. We show that the prion protein contains at least two sequence fragments with highly unusual intrinsic propensities, PrP(114-125) and helix B. No segments with unusual properties were found in Doppel protein, which is topologically identical to PrP but does not undergo structural rearrangements. Known disease-promoting PrP mutations form a statistically significant cluster in the region comprising helices B and C. Due to their unusual properties, PrP(114-125) and the C terminus of helix B may be considered as primary candidates for sites involved in conformational transition from PrPC to PrPSc. The results of our study also show that most PrP mutations associated with neurodegenerative disorders increase local hydrophobicity. We suggest that the observed increase in hydrophobicity may facilitate PrP-to-PrP or/and PrP-to-cofactor interactions, and thus promote structural conversion.     

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).