Expression of truncated PrP targeted to Purkinje cells of PrP knockout mice causes Purkinje cell death and ataxia

PrP knockout mice with disruption of only the PrP-encoding region (Zürich I-type) remain healthy, whereas mice with deletions extending upstream of the PrP-encoding exon (Nagasaki-type) suffer Purkinje cell loss and ataxia, associated with ectopic expression of Doppel in brain, particularly in Purkinje cells. The phenotype is abrogated by co-expression of full-length PrP. Doppel is 25% similar to PrP, has the same globular fold, but lacks the flexible N-terminal tail. We now show that in Zürich I-type PrP-null mice, expression of N-terminally truncated PrP targeted to Purkinje cells also leads to Purkinje cell loss and ataxia, which are reversed by PrP. Doppel and truncated PrP probably cause Purkinje cell degeneration by the same mechanism.     

Doppel蛋白及其对动物生殖的影响

Doppel(简称Dpl)是新发现的一种糖基磷脂酰肌醇锚定(GPI)结构糖蛋白, 在结构上与朊蛋白(Prion protein, PrP)相似, 其编码基因位于朊蛋白编码基因(Prion protein gene, PRNP)下游, 但在生理功能上两者差异较大。Dpl蛋白在成年动物体内的表达主要集中在睾丸组织, 对雄性动物精子完整性、活力以及维持正常受精能力等生殖功能具有重要作用。以下主要综述了Dpl蛋白的生物学特征、生理功能以及对雄性动物生殖调控的影响, 旨在为Dpl蛋白的功能研究和雄性动物生殖调控研究提供理论参考。     

Doppel is an N-glycosylated, glycosylphosphatidylinositol-anchored protein. Expression in testis and ectopic production in the brains of Prnp(0/0) mice predisposed to Purkinje cell loss

The Prnd gene encodes a homolog of the cellular prion protein (PrP(C)) called doppel (Dpl). Up-regulation of Prnd mRNA in two distinct lines of PrP gene ablated (Prnp(0/0)) mice, designated Rcm0 and Ngsk, is associated with death of Purkinje cells. Using recombinant Dpl expressed in Escherichia coli and mouse neuroblastoma cells we demonstrate that wild type (wt) Dpl, like PrP(C), adopts a predominantly alpha-helical conformation, forms intramolecular disulfide bonds, has two N-linked oligosaccharides, and is presented on the cell surface via a glycosylphosphatidylinositol anchor. Dpl protein was detected in testis of wt mice. Using Triton X-114 phase partitioning to enrich for glycosylphosphatidylinositol-anchored proteins, Dpl was detected in brain samples from Rcm0 Prnp(0/0) mice but was absent in equivalent samples from wt mice and ZrchI Prnp(0/0) mice, indicating that ectopic expression of this protein may cause cerebellar pathology in Rcm0 mice. Biochemical and structural similarities between PrP(C) and Dpl documented here parallel the observation that ataxic Ngsk Prnp(0/0) mice can be rescued by overexpression of wild-type PrP transgenes, and suggest that cell surface PrP(C) can antagonize the toxic effect of Dpl expressed in the central nervous system.     

A cluster of familial Creutzfeldt-Jakob disease mutations recapitulate conserved residues in Doppel: a case of molecular mimicry?

Intrachromosomal deletions linking Dpl expression to the PrP promoter produce cerebellar degeneration that can be abrogated by the introduction of wild-type PrP transgenes. Since Dpl-like truncated forms of PrP are neuropathogenic in mice and likewise counterbalanced by expression of PrP(C) we asked whether naturally occurring mutant forms of human PrP have Dpl-like attributes. Five PRNP missense mutations causing familial Creutzfeldt-Jakob disease (F-CJD) map to a helical region found in both PrP(C) and Dpl and result in amino acids identical to conserved residues in Dpl. These F-CJD alleles may cause mutant PrP to become a weak mimetic of Dpl structure and/or function.     

Genomic characterization of the human prion protein (PrP) gene locus

Prion protein (PrP) is intimately linked with a class of neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). Employing bioinformatics and direct molecular analysis, we demonstrated that the human PrP gene (PRNP) locus, which is situated at Chromosome (Chr) position 20p12-ter, contains three genes within a 55-kb interval: PRNP; DOPPEL or PRND, located 20 kb 3? of PRNP; and a novel gene, designated PRNT, that maps 3 kb 3? to PRND and is transcribed to generate at least three alternatively spliced mRNAs. All three genes of this locus demonstrate low sequence homology, implying that, although they may be evolutionarily related, they are functionally distinct. Analysis of both adult and fetal human tissues confirmed the ubiquitous but variable expression profile of PRNP, with the highest levels observed in the CNS and testis. Contrastingly, although PRND shows a wide tissue expression pattern in fetal tissues, it is expressed exclusively in adult testis, whereas all three PRNT isoforms were detected only in adult testis, implying that PRND is developmentally regulated. An investigation of the regulatory mechanisms underlying this complex gene expression pattern from the PRNP locus should provide insight into the function of these genes and the possible involvement of the non-PrP proteins in the development of TSEs.     

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.     

Antagonistic roles of the N-terminal domain of prion protein to doppel

Prion protein (PrP)-like molecule, doppel (Dpl), is neurotoxic in mice, causing Purkinje cell degeneration. In contrast, PrP antagonizes Dpl in trans, rescuing mice from Purkinje cell death. We have previously shown that PrP with deletion of the N-terminal residues 23-88 failed to neutralize Dpl in mice, indicating that the N-terminal region, particularly that including residues 23-88, may have trans-protective activity against Dpl. Interestingly, PrP with deletion elongated to residues 121 or 134 in the N-terminal region was shown to be similarly neurotoxic to Dpl, indicating that the PrP C-terminal region may have toxicity which is normally prevented by the N-terminal domain in cis. We recently investigated further roles for the N-terminal region of PrP in antagonistic interactions with Dpl by producing three different types of transgenic mice. These mice expressed PrP with deletion of residues 25-50 or 51-90, or a fusion protein of the N-terminal region of PrP with Dpl. Here, we discuss a possible model for the antagonistic interaction between PrP and Dpl .     

Newly established in vitro system with fluorescent proteins shows that abnormal expression of downstream prion protein-like protein in mice is probably due to functional disconnection between splicing and 3' formation of prion protein pre-mRNA

We and others previously showed that, in some lines of prion protein (PrP)-knockout mice, the downstream PrP-like protein (PrPLP/Dpl) was abnormally expressed in brains partly due to impaired cleavage/polyadenylation of the residual PrP promoter-driven pre-mRNA despite the presence of a poly(A) signal. In this study, we newly established an in vitro transient transfection system in which abnormal expression of PrPLP/Dpl can be visualized by expression of the green fluorescence protein, EGFP, in cultured cells. No EGFP was detected in cells transfected by a vector carrying a PrP genomic fragment including the region targeted in the knockout mice intact upstream of the PrPLP/Dpl gene. In contrast, deletion of the targeted region from the vector caused expression of EGFP. By employing this system with other vectors carrying various deletions or point mutations in the targeted region, we identified that disruption of the splicing elements in the PrP terminal intron caused the expression of EGFP. Recent lines of evidence indicate that terminal intron splicing and cleavage/polyadenylation of pre-mRNA are functionally linked to each other. Taken together, our newly established system shows that the abnormal expression of PrPLP/Dpl in PrP-knockout mice caused by the impaired cleavage/polyadenylation of the PrP promoter-driven pre-mRNA is due to the functional dissociation between the pre-mRNA machineries, in particular those of cleavage/polyadenylation and splicing. Our newly established in vitro system, in which the functional dissociation between the pre-mRNA machineries can be visualized by EGFP green fluorescence, may be useful for studies of the functional connection of pre-mRNA machineries.     

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.     

Abnormal activation of glial cells in the brains of prion protein-deficient mice ectopically expressing prion protein-like protein, PrPLP/Dpl

BACKGROUND: Some lines of mice homozygous for a disrupted prion protein gene (Prnp), including Ngsk Prnp(0/0) mice, exhibit Purkinje cell degeneration as a consequence of the ectopic overexpression of the downstream gene for prion protein-like protein (PrPLP/Dpl) in the brain, but others, such as Zrch I Prnp(0/0) mice, show neither the neurodegeneration nor the expression of PrPLP/Dpl. In the present study, we found that Ngsk Prnp(0/0), but not Zrch I Prnp(0/0) mice, developed gliosis involving both astrocytes and microglia in the brain. MATERIALS AND METHODS: The brains from wild-type (Prnp(+/+)), Ngsk Prnp(0/0), Zrch I Prnp(0/0), and reconstituted Ngsk Prnp(0/0) mice carrying a mouse PrP transgene, designated Tg(P) Ngsk Prnp(0/0) mice, were subjected into Northern blotting and in situ hybridization using probes of glial fibrillary acidic protein (GFAP) and lysozyme M (LM) specific for astrocytes and microglia, respectively. Immunohistochemistry was also performed on the brain sections using anti-GFAP and anti-F4/80 antibodies. RESULTS: Northern blotting demonstrated upregulated expression of the genes for GFAP and LM in the brains of Ngsk Prnp(0/0), but not in Zrch I Prnp(0/0) mice. A transgene for normal mouse PrP(C) successfully rescued Ngsk Prnp(0/0) mice from the glial activation. In situ hybridization and immunohistochemistry revealed activated astrocytes and microglia mainly in the white matter of both the forebrains and cerebella. In contrast, there was no evidence of neuronal injury except for the Purkinje cell degeneration. Moreover, the glial cell activation was notable well before the onset of the Purkinje cell degeneration. CONCLUSIONS: These findings strongly suggest that ectopic PrPLP/Dpl in the absence of PrP(C) is actively involved in the glial-cell activation in the brain.     

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.     

Biology of the prion gene complex.

The prion protein gene Prnp encodes PrPSc, the major structural component of prions, infectious pathogens causing a number of disorders including scrapie and bovine spongiform encephalopathy (BSE). Missense mutations in the human Prnp gene, PRNP, cause inherited prion diseases such as familial Creutzfeldt-Jakob Disease. In uninfected animals, Prnp encodes a GPI-anchored protein denoted PrPC, and in prion infections, PrPC is converted to PrPSc by templated refolding. Although Prnp is conserved in mammalian species, attempts to verify interactions of putative PrP-binding proteins by genetic means have proven frustrating in that two independent lines of Prnp gene ablated mice (Prnp0/0 mice: ZrchI and Npu) lacking PrPC remain healthy throughout development. This indicates that PrPC serves a function that is not apparent in a laboratory setting or that other molecules have overlapping functions. Shuttling or sequestration of synaptic Cu(II) via binding to N-terminal octapeptide residues and (or) signal transduction involving the fyn kinase are possibilities currently under consideration. A new point of entry into the issue of prion protein function has emerged from identification of a paralog, Prnd, with 25% coding sequence identity to Prnp. Prnd lies downstream of Prnp and encodes the Dpl protein. Like PrPC, 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 two other lines of Prnp0/0 mice (Ngsk and Rcm0) via intergenic splicing events. These lines of Prnp0/0 mice exhibit ataxia and apoptosis of cerebellar cells, indicating that ectopic synthesis of Dpl protein is toxic to CNS 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 wt Prnp transgenes. The interaction between the Prnp and Prnd genes in mouse cerebellar neurons may have a physical correlate in competition between Dpl and PrPC within a common biochemical pathway that, when misregulated, leads to apoptosis.     

Copper(II) binding to the human Doppel protein may mark its functional diversity from the prion protein

Doppel (Dpl) is the first described homologue of the prion protein, the main constituent of the agent responsible for prion diseases. The cellular prion protein (PrP(C)) is predominantly present in the central nervous system. Although its role is not yet completely clarified, PrP(C) seems to be involved in Cu(2+) recycling from synaptic clefts and in preventing neuronal oxidative damage. Conversely, Dpl is expressed in heart and testis and has been shown to regulate male fertility by intervening in gametogenesis and sperm-egg interactions. Therefore, despite a high sequence homology and a similar three-dimensional fold, the functions of PrP(C) and Dpl appear unrelated. Here we show by electron paramagnetic resonance and fluorescence spectroscopy that the in vitro binding of copper(II) to human recombinant Dpl occurs with a different pattern from that observed for recombinant PrP. At physiological pH values, two copper(II)-binding sites with different affinities were found in Dpl. At lower pH values, two additional copper(II)-binding sites can be identified as follows: one complex is present only at pH 4, and the other is observed in the pH range 5-6. As derived from the electron paramagnetic resonance characteristics, all Dpl-copper(II) complexes have a different coordination sphere from those present in PrP. Furthermore, in contrast to the effect shown previously for PrP(C), addition of Cu(2+) to Dpl-expressing cells does not cause Dpl internalization. These results suggest that binding of the ion to PrP(C) and Dpl may contribute to the different functional roles ascribed to these highly homologous proteins.     

Bovine prion (PrP) and Doppel (Dpl) proteins expression after in vitro leukocyte activation or Dpl/PrP blocking

It has been postulated that Doppel (Dpl) and Prion (PrP) proteins have yet undetermined interactions, since Dpl is overexpressed in transgenic PrP-deficient mice. In this study we investigated the expression levels of Dpl and PrP on lymphocytes, monocytes and neutrophils (PMNs) isolated from bovine blood and incubated (2 and 18 h) with TNFalpha, IL-1, IL-2, IL-8, C5a, IFNgamma, anti-PrP, and anti-Dpl antibodies by flow cytometry. The isolation procedures yielded cell populations with high purity, viability and recovery rates. After 2 h incubation, expression of PrP or Dpl was altered only in PMNs. These cells overexpressed PrP when incubated with TNFalpha and IFNgamma, and both PrP and Dpl when incubated with C5a; incubation with TNFalpha, IL-8 and IFNgamma led to down-regulation of Dpl. Lymphocytes incubated for 18 h with IL-2 and with IFNgamma overexpressed Dpl. Incubation with the anti-PrP antibody induced down-regulation of Dpl in PMNs after 2 h and overexpression of Dpl in lymphocytes after 18 h. The differences recorded after 2 h were likely due to redistribution of pre-existing PrP or Dpl molecules, while those seen at 18 h were most probably due to increased protein synthesis. The variations seen using the different activators depend on different receptors and/or signaling pathways. These results demonstrate that is possible to alter the expression of Dpl and PrP in blood cells in vitro by incubation with either cytokines or anti-PrP antibodies. This opens an interesting opportunity to study the biology of these proteins using in vitro systems.     

Absence of the prion protein homologue Doppel causes male sterility

The agent that causes prion diseases is thought to be identical with PrP(Sc), a conformer of the normal prion protein PrP(C). PrP(C)-deficient mice do not exhibit major pathologies, perhaps because they express a protein termed Dpl, which shares significant biochemical and structural homology with PrP(C). To investigate the physiological function of Dpl, we generated mice harbouring a homozygous disruption of the Prnd gene that encodes Dpl. Dpl deficiency did not interfere with embryonic and postnatal development, but resulted in male sterility. Dpl protein was expressed at late stages of spermiogenesis, and spermatids of Dpl mutants were reduced in numbers, immobile, malformed and unable to fertilize oocytes in vitro. Mechanical dissection of the zona pellucida partially restored in vitro fertilization. We conclude that Dpl regulates male fertility by controlling several aspects of male gametogenesis and sperm-egg interaction.     

The role of Bax and caspase-3 in doppel-induced apoptosis of cerebellar granule cells

Doppel (Dpl) protein is a paralog of the prion protein (PrP) that shares 25% sequence similarity with the C-terminus of PrP, a common N-glycosylation site and a C-terminal signal peptide for attachment of a glycosylphophatidyl inositol anchor. Whereas PrP^C is highly expressed in the central nervous system (CNS), Dpl is detected mostly in testes and its ectopic expression in the CNS leads to ataxia as well as Purkinje and granule cell degeneration in the cerebellum. The mechanism through which Dpl induces neurotoxicity is still debated. In the present work, primary neuronal cultures derived from postnatal cerebellar granule cells of wild-type and PrP-knockout FVB mice were used in order to investigate the molecular events that occur upon exposure to Dpl. Treatment of cultured cerebellar neurons with recombinant Dpl produced apoptosis that could be prevented by PrP co-incubation. When primary neuronal cultures from Bax-deficient mice were incubated with Dpl, no apoptosis was observed, suggesting an important role of Bax in triggering neurodegeneration. Similarly, cell survival increased when recDpl-treated cells were incubated with an inhibitor of caspase-3, which mediates apoptosis in mammalian cells. Together, our findings raise the possibility that Bax and caspase-3 feature in Dpl-mediated apoptosis.     

The role of Bax and caspase-3 in doppel-induced apoptosis of cerebellar granule cells

Doppel (Dpl) protein is a paralog of the prion protein (PrP) that shares 25% sequence similarity with the C-terminus of PrP, a common N-glycosylation site and a C-terminal signal peptide for attachment of a glycosylphophatidyl inositol anchor. Whereas PrP^C is highly expressed in the central nervous system (CNS), Dpl is detected mostly in testes and its ectopic expression in the CNS leads to ataxia as well as Purkinje and granule cell degeneration in the cerebellum. The mechanism through which Dpl induces neurotoxicity is still debated. In the present work, primary neuronal cultures derived from postnatal cerebellar granule cells of wild-type and PrP-knockout FVB mice were used in order to investigate the molecular events that occur upon exposure to Dpl. Treatment of cultured cerebellar neurons with recombinant Dpl produced apoptosis that could be prevented by PrP co-incubation. When primary neuronal cultures from Bax-deficient mice were incubated with Dpl, no apoptosis was observed, suggesting an important role of Bax in triggering neurodegeneration. Similarly, cell survival increased when recDpl-treated cells were incubated with an inhibitor of caspase-3, which mediates apoptosis in mammalian cells. Together, our findings raise the possibility that Bax and caspase-3 feature in Dpl-mediated apoptosis.