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.     

Deletion of N-terminal residues 23-88 from prion protein (PrP) abrogates the potential to rescue PrP-deficient mice from PrP-like protein/doppel-induced Neurodegeneration

Accumulating evidence has suggested that prion protein (PrP) is neuroprotective and that a PrP-like protein/Doppel (PrPLP/Dpl) is neurotoxic. A line of PrP-deficient mice, Ngsk Prnp0/0, ectopically expressing PrPLP/Dpl in neurons, exhibits late-onset ataxia because of Purkinje cell death that is prevented by a transgene encoding wild-type mouse PrP. To elucidate the mechanisms of neurodegeneration in these mice, we introduced five types of PrP transgene, namely one heterologous hamster, two mouse/hamster chimeric genes, and two mutants, each of which encoded PrP lacking residues 23-88 (MHM2.del23-88) or with E199K substitution (Mo.E199K), into Ngsk Prnp0/0 mice. Only MHM2.del23-88 failed to rescue the mice from the Purkinje cell death. The transgenic mice, MHM2.del23-88/Ngsk Prnp0/0, expressed several times more PrP than did wild-type (Prnp+/+) mice and PrPLP/Dpl at an equivalent level to Ngsk Prnp0/0 mice. Little difference was observed in the pathology and onset of ataxia between Ngsk Prnp0/0 and MHM2.del23-88/Ngsk Prnp0/0. No detergent-insoluble PrPLP/Dpl was detectable in the central nervous system of Ngsk Prnp0/0 mice even after the onset of ataxia. Our findings provide evidence that the N-terminal residues 23-88 of PrP containing the unique octapeptide-repeat region is crucial for preventing Purkinje cell death in Prnp0/0 mice expressing PrPLP/Dpl in the neuron.     

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.     

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.     

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.     

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.     

Age-Dependent Impairment of Eyeblink Conditioning in Prion Protein-Deficient Mice

Mice lacking the prion protein (PrP^C) gene (Prnp), Ngsk Prnp ^0/0 mice, show late-onset cerebellar Purkinje cell (PC) degeneration because of ectopic overexpression of PrP^C-like protein (PrPLP/Dpl). Because PrP^C is highly expressed in cerebellar neurons (including PCs and granule cells), it may be involved in cerebellar synaptic function and cerebellar cognitive function. However, no studies have been conducted to investigate the possible involvement of PrP^C and/or PrPLP/Dpl in cerebellum-dependent discrete motor learning. Therefore, the present cross-sectional study was designed to examine cerebellum-dependent delay eyeblink conditioning in Ngsk Prnp ^0/0 mice in adulthood (16, 40, and 60 weeks of age). The aims of the present study were two-fold: (1) to examine the role of PrP^C and/or PrPLP/Dpl in cerebellum-dependent motor learning and (2) to confirm the age-related deterioration of eyeblink conditioning in Ngsk Prnp ^0/0 mice as an animal model of progressive cerebellar degeneration. Ngsk Prnp ^0/0 mice aged 16 weeks exhibited intact acquisition of conditioned eyeblink responses (CRs), although the CR timing was altered. The same result was observed in another line of PrP^c-deficient mice, ZrchI PrnP ^0/0 mice. However, at 40 weeks of age, CR incidence impairment was observed in Ngsk Prnp ^0/0 mice. Furthermore, Ngsk Prnp ^0/0 mice aged 60 weeks showed more significantly impaired CR acquisition than Ngsk Prnp ^0/0 mice aged 40 weeks, indicating the temporal correlation between cerebellar PC degeneration and motor learning deficits. Our findings indicate the importance of the cerebellar cortex in delay eyeblink conditioning and suggest an important physiological role of prion protein in cerebellar motor learning.     

BCL-2 counteracts Doppel-induced apoptosis of prion-protein-deficient Purkinje cells in the Ngsk Prnp(0/0) mouse

The pro-apoptotic factor BAX has recently been shown to contribute to Purkinje cell (PC) apoptosis induced by the neurotoxic prion-like protein Doppel (Dpl) in the prion-protein-deficient Ngsk Prnp(0/0) (NP(0/0)) mouse. In view of cellular prion protein (PrP(c)) ability to counteract Dpl neurotoxicity and favor neuronal survival like BCL-2, we investigated the effects of the anti-apoptotic factor BCL-2 on Dpl neurotoxicity by studying the progression of PC death in aging NP(0/0)-Hu-bcl-2 double mutant mice overexpressing human BCL-2 (Hu-bcl-2). Quantitative analysis showed that significantly more PCs survived in NP(0/0)-Hu-bcl-2 double mutants compared with the NP(0/0) mutants. However, number of PCs remained inferior to wild-type levels and to the increased number of PCs observed in Hu-bcl-2 mutants. In the NP(0/0) mutants, Dpl-induced PC death occurred preferentially in the aldolase C-negative parasagittal compartments of the cerebellar cortex. Activation of glial cells exclusively in these compartments, which was abolished by the expression of Hu-bcl-2 in the double mutants, suggested that chronic inflammation is an indirect consequence of Dpl-induced PC death. This partial rescue of NP(0/0) PCs by Hu-bcl-2 expression was similar to that observed in NP(0/0):Bax(-/-) double mutants with bax deletion. Taken together, these data strongly support the involvement of BCL-2 family-dependent apoptotic pathways in Dpl neurotoxicity. The capacity of BCL-2 to compensate PrP(c) deficiency by rescuing PCs from Dpl-induced death suggests that the BCL-2-like property of PrP(c) may impair Dpl-like neurotoxic pathways in wild-type neurons.     

Prion protein suppresses perturbation of cellular copper homeostasis under oxidative conditions

Prion protein (PrP) binds copper and exhibits superoxide dismutase-like activity, while the roles of PrP in copper homeostasis remain controversial. Using Zeeman graphite furnace atomic absorption spectroscopy, we quantified copper levels in immortalized PrP gene (Prnp)-deficient neuronal cells transfected with Prnp and/or Prnd, which encodes PrP-like protein (PrPLP/Dpl), in the presence or absence of oxidative stress induced by serum deprivation. In the presence of serum, copper levels were not significantly affected by the expression of PrP and/or PrPLP/Dpl, whereas serum deprivation induced a decrease in copper levels that was inhibited by PrP but not by PrPLP/Dpl. The inhibitory effect of PrP on the decrease of copper levels was prevented by overexpression of PrPLP/Dpl. These findings indicate that PrP specifically stabilizes copper homeostasis, which is perturbed under oxidative conditions, while PrPLP/Dpl overexpression prevents PrP function in copper homeostasis, suggesting an interaction of PrP and PrPLP/Dpl and distinct functions between PrP and PrPLP/Dpl on metal homeostasis. Taken together, these results strongly suggest that PrP, in addition to its antioxidant properties, plays a role in stabilizing cellular copper homeostasis under oxidative conditions.     

Loss of the cellular prion protein affects the Ca2+ homeostasis in hippocampal CA1 neurons

Previous neurophysiological studies on prion protein deficient (Prnp(-/-)) mice have revealed a significant reduction of slow afterhyperpolarization currents (sI(AHP)) in hippocampal CA1 pyramidal cells. Here we aim to determine whether loss of PrP(C.) directly affects the potassium channels underlying sI(AHP) or if sI(AHP) is indirectly disturbed by altered intracellular Ca(2+) fluxes. Patch-clamp measurements and confocal Ca(2+) imaging in acute hippocampal slice preparations of Prnp(-/-) mice compared to littermate control mice revealed a reduced Ca(2+) rise in CA1 neurons lacking PrP(C) following a depolarization protocol known to induce sI(AHP). Moreover, we observed a reduced Ca(2+) influx via l-type voltage gated calcium channels (VGCCs). No differences were observed in the protein expression of the pore forming alpha1 subunit of VGCCs Prnp(-/-) mice. Surprisingly, the beta2 subunit, critically involved in the transport of the alpha1 subunit to the plasma membrane, was found to be up-regulated in knock out hippocampal tissue. On mRNA level however, no differences could be detected for the alpha1C, D and beta2-4 subunits. In conclusion our data support the notion that lack of PrP(C.) does not directly affect the potassium channels underlying sI(AHP), but modulates these channels due to its effect on the intracellular free Ca(2+) concentration via a reduced Ca(2+) influx through l-type VGCCs.     

Impairment of superoxide dismutase activation by N-terminally truncated prion protein (PrP) in PrP-deficient neuronal cell line

Previous studies have reported a neuroprotective role for cellular prion protein (PrP(C)) against apoptosis induced by serum deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line, but the mechanisms remain unclear. In this study, to investigate the mechanisms by which PrP(C) prevents apoptosis, the authors compared apoptosis of Prnp(-/-) cells with that of Prnp(-/-) cells expressing the wild-type PrP(C) or PrP(C) lacking N-terminal octapeptide repeat region under serum-free conditions. Re-introduction of Prnp rescued cells from apoptosis, upregulated superoxide dismutase (SOD) activity, enhanced superoxide anion elimination, and inhibited caspase-3/9 activation. On the other hand, N-terminally truncated PrP(C) enhanced apoptosis accompanied by potentiation of superoxide production and caspase-3/9 activation due to inhibition of SOD. These results suggest that PrP(C) protects Prnp(-/-) cells from apoptosis via superoxide- and caspase-3/9-dependent pathways by upregulating SOD activity. Furthermore, the octapeptide repeat region of PrP(C) plays an essential role in regulating apoptosis and SOD activity.     

BAX contributes to Doppel-induced apoptosis of prion-protein-deficient Purkinje cells

Research efforts to deduce the function of the prion protein (PrPc) in knock-out mouse mutants have revealed that large deletions in the PrPc genome result in the ectopic neuronal expression of the prion-like protein Doppel (Dpl). In our analysis of one such line of mutant mice, Ngsk Prnp0/0 (NP0/0), we demonstrate that the ectopic expression of Dpl in brain neurons induces significant levels of cerebellar Purkinje cell (PC) death as early as six months after birth. To investigate the involvement of the mitochondrial proapoptotic factor BAX in the Dpl-induced apoptosis of PCs, we have analyzed the progression of PC death in aging NP0/0:Bax-/- double knockout mutants. Quantitative analysis of cell numbers showed that significantly more PCs survived in NP0/0:Bax-/- double mutants than in the NP0/0:Bax+/+ mutants. However, PC numbers were not restored to wildtype levels or to the increased number of PCs observed in Bax-/- mutants. The partial rescue of NP0/0 PCs suggests that the ectopic expression of Dpl induces both BAX-dependent and BAX-independent pathways of cell death. The activation of glial cells that is shown to be associated topographically with Dpl-induced PC death in the NP0/0:Bax+/+ mutants is abolished by the loss of Bax expression in the double mutant mice, suggesting that chronic inflammation is an indirect consequence of Dpl-induced PC death.     

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.     

PrP mRNA and protein expression in brain and PrPc in CSF in Creutzfeldt-Jakob disease MM1 and VV2

Creutzfeldt-Jakob disease (CJD) is a heterogenic neurodegenerative disorder associated with abnormal post-translational processing of cellular prion protein (PrP^c). CJD displays distinctive clinical and pathological features which correlate with the genotype at the codon 129 (methionine or valine: M or V respectively) in the prion protein gene and with size of the protease-resistant core of the abnormal prion protein PrP^sc (type 1: 20/21 kDa and type 2: 19 kDa). MM1 and VV2 are the most common sporadic CJD (sCJD) subtypes. PrP mRNA expression levels in the frontal cortex and cerebellum are reduced in sCJD in a form subtype-dependent. Total PrP protein levels and PrP^sc levels in the frontal cortex and cerebellum accumulate differentially in sCJD MM1 and sCJD VV2 with no relation between PrP^sc deposition and spongiform degeneration and neuron loss, but with microgliosis, and IL6 and TNF-α response. In the CSF, reduced PrP^c, the only form present in this compartment, occurs in sCJD MM1 and VV2. PrP mRNA expression is also reduced in the frontal cortex in advanced stages of Alzheimer disease, Lewy body disease, progressive supranuclear palsy, and frontotemporal lobe degeneration, but PrP^c levels in brain varies from one disease to another. Reduced PrP^c levels in CSF correlate with PrP mRNA expression in brain, which in turn reflects severity of degeneration in sCJD.     

No superoxide dismutase activity of cellular prion protein in vivo

Prion diseases are characterized by the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(C), which is encoded by the Prnp gene. PrP(C) is highly expressed on neurons and its function is unknown. Recombinant PrP(C) was claimed to possess superoxide dismutase (SOD) activity, and it was hypothesized that abrogation of this function may contribute to neurodegeneration in prion diseases. We tested this hypothesis in vivo by studying copper/zinc and manganese SOD activity in genetically defined crosses of mice lacking the Sod1 gene with mice lacking PrP(C), and with hemizygous or homozygous tga20 transgenic mice overexpressing various levels of PrP(C). We failed to detect any influence of the Prnp genotype and gene dosage on SOD1 or SOD2 activity in heart, spleen, brain, and synaptosome-enriched brain fractions. Control experiments included crosses of mice lacking or overexpressing PrPc with mice overexpressing human Cu2+/Zn2+-superoxide dismutase, and confirmed that SOD enzymatic activity correlated exclusively with the gene dosage of bona fide human or murine SOD. We conclude that PrP(C) in vivo does not discernibly contribute to total SOD activity and does not possess an intrinsic dismutase activity.     

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.     

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.     

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.