Regulation of GABAA and Glutamate Receptor Expression,Synaptic Facilitation and Long-Term Potentiation in the Hippocampus of Prion Mutant Mice

Background

Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrP^sc) of the natural cellular prion protein (PrP^c) encoded by the Prnp gene. Although several roles have been attributed to PrP^c, its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrP^c studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation.

Methodology/Principal Findings

Here we explore the role of PrP^c expression in neurotransmission and neural excitability using wild-type, Prnp −/− and PrP^c-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp −/− mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina™ microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp −/− and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABA_A and AMPA-kainate receptors are co-regulated in both Prnp −/− and Tg20 mice.

Conclusions/Significance

Present results demonstrate that PrP^c is necessary for the proper homeostatic functioning of hippocampal circuits, because of its relationships with GABA_A and AMPA-Kainate neurotransmission. New PrP^c functions have recently been described, which point to PrP^c as a target for putative therapies in Alzheimer''s disease. However, our results indicate that a “gain of function” strategy in Alzheimer''s disease, or a “loss of function” in prionopathies, may impair PrP^c function, with devastating effects. In conclusion, we believe that present data should be taken into account in the development of future therapies.     

Oxidative stress impairs autophagic flux in prion protein-deficient hippocampal cells

We previously reported that autophagy is upregulated in Prnp-deficient (Prnp^0/0) hippocampal neuronal cells in comparison to cellular prion protein (PrP^C)-expressing (Prnp^+/+) control cells under conditions of serum deprivation. In this study, we determined whether a protective mechanism of PrP^C is associated with autophagy using Prnp^0/0 hippocampal neuronal cells under hydrogen peroxide (H₂O₂)-induced oxidative stress. We found that Prnp^0/0 cells were more susceptible to oxidative stress than Prnp^+/+ cells in a dose- and time-dependent manner. In addition, we observed enhanced autophagy by immunoblotting, which detected the conversion of microtubule-associated protein 1 light chain 3 β (LC3B)-I to LC3B-II, and we observed increased punctate LC3B immunostaining in H₂O₂-treated Prnp^0/0 cells compared with H₂O₂-treated control cells. Interestingly, this enhanced autophagy was due to impaired autophagic flux in the H₂O₂-treated Prnp^0/0 cells, while the H₂O₂-treated Prnp^+/+ cells showed enhanced autophagic flux. Furthermore, caspase-dependent and independent apoptosis was observed when both cell lines were exposed to H₂O₂. Moreover, the inhibition of autophagosome formation by Atg7 siRNA revealed that increased autophagic flux in Prnp^+/+ cells contributes to the prosurvival effect of autophagy against H₂O₂ cytotoxicity. Taken together, our results provide the first experimental evidence that the deficiency of PrP^C may impair autophagic flux via H₂O₂-induced oxidative stress.     

Unraveling the neuroprotective mechanisms of PrPC in excitotoxicity

Knowledge of the natural roles of cellular prion protein (PrP^C) is essential to an understanding of the molecular basis of prion pathologies. This GPI-anchored protein has been described in synaptic contacts, and loss of its synaptic function in complex systems may contribute to the synaptic loss and neuronal degeneration observed in prionopathy. In addition, Prnp knockout mice show enhanced susceptibility to several excitotoxic insults, GABA_A receptor-mediated fast inhibition was weakened, LTP was modified and cellular stress increased. Although little is known about how PrP^C exerts its function at the synapse or the downstream events leading to PrP^C-mediated neuroprotection against excitotoxic insults, PrP^C has recently been reported to interact with two glutamate receptor subunits (NR2D and GluR6/7). In both cases the presence of PrP^C blocks the neurotoxicity induced by NMDA and Kainate respectively. Furthermore, signals for seizure and neuronal cell death in response to Kainate in Prnp knockout mouse are associated with JNK3 activity, through enhancing the interaction of GluR6 with PSD-95. In combination with previous data, these results shed light on the molecular mechanisms behind the role of PrP^C in excitotoxicity. Future experimental approaches are suggested and discussed.     

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.     

Implications of prion polymorphisms

The sequence of a host’s prion protein (PrP) can affect that host’s susceptibility to prion disease and is the primary basis for the species barrier to transmission. Yet within many species, polymorphisms of the prion protein gene (Prnp) exist, each of which can further affect susceptibility or influence incubation period, pathology and phenotype. As strains are defined by these features (incubation period, pathology, phenotype), polymorphisms may also lead to the preferential propagation or generation of certain strains. In our recent study of the mouse Prnp^a and Prnp^b polymorphisms (which produced the proteins PrP^a and PrP^b, respectively), we found differences in aggregation tendency, strain adaptability and conformational variability. Comparing our in vitro data with that of in vivo studies, we found that differing incubation periods between Prnp^a and Prnp^b mice can primarily be explained on the basis of faster or more efficient aggregation of PrP^a. In addition, and more importantly, we found that the faithful propagation of strains in Prnp^b mice can be explained by the ability of PrP^b to adopt a wider range of conformations. This adaptability allows PrP^b to successfully propagate the structural features of a seed. In contrast, Prnp^a mice revert PrP^b strains into PrP^a -type strains, and overall they have a narrower distribution of incubation periods. This can be explained by PrP^a having fewer preferred conformations. We propose that Prnp polymorphisms are one route by which certain prion strains may preferentially propagate. This has significant implications for prion disease, chronic wasting disease (CWD) in particular, as it is spreading through North America. Deer which are susceptible to CWD also carry polymorphisms which influence their susceptibility. If these polymorphisms also preferentially allow strain diversification and propagation, this may accelerate the crossing of species barriers and propagation of the disease up the food chain.     

The Neuroprotective Effect of Curcumin and Nigella sativa Oil Against Oxidative Stress in the Pilocarpine Model of Epilepsy: A Comparison with Valproate

Oxidative stress has been implicated to play a role in epileptogenesis and pilocarpine-induced seizures. The present study aims to evaluate the antioxidant effects of curcumin, Nigella sativa oil (NSO) and valproate on the levels of malondialdehyde, nitric oxide, reduced glutathione and the activities of catalase, Na⁺, K⁺-ATPase and acetylcholinesterase in the hippocampus of pilocarpine-treated rats. The animal model of epilepsy was induced by pilocarpine and left for 22 days to establish the chronic phase of epilepsy. These animals were then treated with curcumin, NSO or valproate for 21 days. The data revealed evidence of oxidative stress in the hippocampus of pilocarpinized rats as indicated by the increased nitric oxide levels and the decreased glutathione levels and catalase activity. Moreover, a decrease in Na⁺, K⁺-ATPase activity and an increase in acetylcholinesterase activity occurred in the hippocampus after pilocarpine. Treatment with curcumin, NSO or valproate ameliorated most of the changes induced by pilocarpine and restored Na⁺, K⁺-ATPase activity in the hippocampus to control levels. This study reflects the promising anticonvulsant and potent antioxidant effects of curcumin and NSO in reducing oxidative stress, excitability and the induction of seizures in epileptic animals and improving some of the adverse effects of antiepileptic drugs.     

Tissue- and cell type-specific modification of prion protein (PrP)-like protein Doppel, which affects PrP endoproteolysis

A prion protein (PrP)-like protein, Doppel (Dpl) is a homologue of cellular PrP (PrP^C). Immunoblotting revealed heterogeneous glycosylation patterns of Dpl and PrP^C in several cell lines and tissues, including brain and testis. To investigate whether the glycosylation and modification of Dpl and PrP^C could influence each other, PrP gene (Prnp)-deficient neuronal cells, transfected with Prnp and/or the Dpl gene (Prnd), were analyzed by deglycosylation with peptide N-glycosidase F. The modification of Dpl was not influenced by PrP^C, whereas an N-terminally truncated fragment of PrP^C was reduced by Dpl expression. These results indicated that Dpl was glycosylated in a cell type- and tissue-specific manner regardless of PrP^C, while PrP^C endoproteolysis was modulated by Dpl expression.     

Unraveling the neuroprotective mechanisms of PrPC in excitotoxicity

Knowledge of the natural roles of cellular prion protein (PrP^C) is essential to an understanding of the molecular basis of prion pathologies. This GPI-anchored protein has been described in synaptic contacts, and loss of its synaptic function in complex systems may contribute to the synaptic loss and neuronal degeneration observed in prionopathy. In addition, Prnp knockout mice show enhanced susceptibility to several excitotoxic insults, GABA_A receptor-mediated fast inhibition was weakened, LTP was modified and cellular stress increased. Although little is known about how PrP^C exerts its function at the synapse or the downstream events leading to PrP^C-mediated neuroprotection against excitotoxic insults, PrP^C has recently been reported to interact with two glutamate receptor subunits (NR2D and GluR6/7). In both cases the presence of PrP^C blocks the neurotoxicity induced by NMDA and Kainate respectively. Furthermore, signals for seizure and neuronal cell death in response to Kainate in Prnp knockout mouse are associated with JNK3 activity, through enhancing the interaction of GluR6 with PSD-95. In combination with previous data, these results shed light on the molecular mechanisms behind the role of PrP^C in excitotoxicity. Future experimental approaches are suggested and discussed.     

Establishment of a new glial cell line from hippocampus of prion protein gene-deficient mice

Cellular prion protein (PrP^C) is expressed not only in neuronal cells but also in non-neuronal cells such as astroglial cells. In the present study, the prion protein (PrP) gene (Prnp)-deficient astroglial cell line GpL1 from hippocampal cells of ZrchI Prnp^−/− mice were established. Transfection of Prnp suppressed cell death in GpL1 cells under serum-free conditions. The PrP-expressing GpL1 cells showed increased superoxide dismutase activity compared to control GpL1 cells. These results suggest that the anti-oxidative activity of PrP^C functions in not only neuronal cells but also astroglial cells possibly due to the increased anti-oxidative activity of astroglial cells.     

Identification and characterization of a novel mouse prion gene allele

The major determinant of prion disease incubation time in mice is thought to be the amino acid sequence of the prion protein. Two alleles of the mouse prion gene (Prnp) have been described, where Prnp^a (Leu-108, Thr-189) and Prnp^b (Phe-108, Val-189) are associated with short and long incubation times, to defined prion strains, respectively. As part of a survey of inbred mouse lines, the prion gene open reading frame was sequenced and revealed a new allele, Prnp^c (Phe-108, Thr-189), in the strain MAI/Pas. To study the influence of Prnp^c independently of the MAI/Pas genetic background, we generated a congenic line in which Prnp^c was bred onto the C57BL/6JOlaHsd background. Following intracerebral inoculation with Chandler/RML scrapie prions, the congenic mice showed an increased mean incubation time relative to C57BL/6JOlaHsd, of over 100 days. However, no differences were observed in the intensity and pattern of PrP immunoreactivity deposition or spongiosis. We conclude that the new allele, Prnp^c, modulates incubation time but not neuropathology and that the previous classification of mice into two distinct groups based on incubation time and Prnp genotype should now be revised.     

Mouse-Hamster Chimeric Prion Protein (PrP) Devoid of N-Terminal Residues 23-88 Restores Susceptibility to 22L Prions,but Not to RML Prions in PrP-Knockout Mice

Prion infection induces conformational conversion of the normal prion protein PrP^C, into the pathogenic isoform PrP^Sc, in prion diseases. It has been shown that PrP-knockout (Prnp^0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp^0/0 mice, neither developed the disease nor accumulated MHM2^ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice developed the disease with abundant accumulation of MHM2^ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2^ScΔ23-88, and that the co-expressing wild-type PrP^C can stimulate the conversion of MHM2Δ23-88 to MHM2^ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp^0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp^0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2^ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2^ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice. However, wild-type PrP^Sc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp^0/+ mice, compared with RML- and 22L-inoculated Prnp^0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2^ScΔ23-88 without the help of the trans-acting PrP^C, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrP^C stimulates the conversion of MHM2Δ23-88 into MHM2^ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrP^C into PrP^Sc.     

Neocortex- and hippocampus-specific deletion of Gabrg2 causes temperature-dependent seizures in mice

Mutations in the GABRG2 gene encoding the γ-aminobutyric acid (GABA) A receptor gamma 2 subunit are associated with genetic epilepsy with febrile seizures plus, febrile seizures plus, febrile seizures, and other symptoms of epilepsy. However, the mechanisms underlying Gabrg2-mediated febrile seizures are poorly understood. Here, we used the Cre/loxP system to generate conditional knockout (CKO) mice with deficient Gabrg2 in the hippocampus and neocortex. Heterozygous CKO mice (Gabrg2^fl/wtCre⁺) exhibited temperature-dependent myoclonic jerks, generalised tonic-clonic seizures, increased anxiety-like symptoms, and a predisposition to induce seizures. Cortical electroencephalography showed the hyperexcitability in response to temperature elevation in Gabrg2^fl/wtCre⁺ mice, but not in wild-type mice. Gabrg2^fl/wtCre⁺ mice exhibited spontaneous seizures and susceptibility to temperature-induced seizures. Loss of neurons were observed in cortical layers V–VI and hippocampus of Gabrg2^fl/wtCre⁺ mice. Furthermore, the latency of temperature- or pentylenetetrazol-induced seizures were significantly decreased in Gabrg2^fl/wtCre⁺ mice compared with wild-type mice. In summary, Gabrg2^fl/wtCre⁺ mice with Gabrg2 deletion in the neocortex and hippocampus reproduce many features of febrile seizures and therefore provide a novel model to further understand this syndrome at the cellular and molecular level.Subject terms: Epilepsy, Genetics of the nervous system     

Neuroprotective properties of the PrP-like Shadoo glycoprotein assessed in the middle cerebral artery occlusion model of ischemia

ABSTRACT

Biochemical similarities have been noted between the natively unstructured region of the cellular prion protein, PrP^C, and a GPI-linked glycoprotein called Shadoo (Sho); these proteins are encoded by the Prnp and Sprn genes, respectively. Both proteins are expressed in the adult central nervous system and they share overlapping partners, including each other, in interactome studies. As prior studies have ascribed neuroprotective properties to the N-terminal region of PrP^C, specifically the octarepeat region, we investigated Sho's neuroprotective properties. To this end we assessed Sho-null (Sprn^0/0) and hemizygous (Sprn^0/+) mice in the middle cerebral artery occlusion (MCAO) model versus wild type mice and also vs. transgene-rescued Sprn^0/0-TgSprn mice. Sprn^0/0 mice had a tendency to greater fragility in reaching endpoint and deficits in parameters including infarct volume and neurogenesis, with a reciprocal trend noted in transgene-rescued mice; however these effects did not reach significance. Loss of both PrP^C and Sho immunostaining occurred in parallel to neuronal loss on the ipsilateral side of MCAO-lesioned animals; while focal elevations in immunostaining in the penumbra region were sometimes evident for PrP^C, they were not noted for Sho. Our studies argue against discernible neuroprotective action of Sho in the genetic backgrounds used for this MCAO paradigm. Whether or not the positively charged N-terminal regions in Sho and PrP^C fulfil different roles in vivo remains to be determined.     

Behavioral abnormalities in prion protein knockout mice and the potential relevance of PrPC for the cytoskeleton

The cellular prion protein (PrP^C) is a highly conserved protein, which is anchored to the outer surface of the plasma membrane. Even though its physiological function has already been investigated in different cell or mouse models where PrP^C expression is either upregulated or depleted, its exact physiological role in a mammalian organism remains elusive. Recent studies indicate that PrP^C has multiple functions and is involved in cognition, learning, anxiety, locomotion, depression, offensive aggression and nest building behavior. While young animals (3 months of age) show only marginal abnormalities, most of the deficits become apparent as the animals age, which might indicate its role in neurodegeneration or neuroprotection. However, the exact biochemical mechanism and signal transduction pathways involving PrP^C are only gradually becoming clearer. We report the observations made in different studies using different Prnp0/0 mouse models and propose that PrP^C plays an important role in the regulation of the cytoskeleton and associated proteins. In particular, we showed a nocodazole treatment influenced colocalization of PrP^C and α tubulin 1. In addition, we confirmed the observed deficits in nest building using a different backcrossed Prnp0/0 mouse line.     

Review of studies that have used knockout mice to assess normal function of prion protein under immunological or pathophysiological stress

Deletion of cellular isoform of prion protein (PrP^C) increases neuronal predisposition to damage by modulating apoptosis and the negative consequences of oxidative stress. In vivo studies have demonstrated that PrP^C‐deficient mice are more prone to seizure, depression, and induction of epilepsy and experience extensive cerebral damage following ischemic challenge or viral infection. In addition, adenovirus‐mediated overexpression of PrP^C reduces brain damage in rat models of cerebral ischemia. In experimental autoimmune encephalomyelitis, PrP^C‐deficient mice reportedly have a more aggressive disease onset and less clinical improvement during the chronic phase than wild‐type mice mice. In mice given oral dextran sulfate, PrP^C has a potential protective role against inflammatory bowel disease. PrP^C‐deficient mice demonstrate significantly greater increases in blood glucose concentrations after intraperitoneal injection of glucose than wild‐type mice. Further in vivo challenges to PrP gene‐deficient models and conditional knockout models with siRNA and in vivo administration of PrP‐ligating agents may assist in refining knowledge of the lymphoid function of PrP^C and predicting the effects of anti‐PrP treatment on the immune system. Together, these findings indicate that PrP^C may have multiple neuroprotective and anti‐inflammatory roles, which explains why this protein is so widely expressed.     

Prion Protein Is Necessary for Latent Learning and Long-Term Memory Retention

1. The cellular prion protein, designated PrP^c, is a key molecule in the prion diseases but its physiological function remains unknown. To elucidate whether PrP^c plays some role in the central nervous system, we established a line of mice in which the PrP gene had been disrupted and subsequently conducted long-term observations.2. Performance in latent learning and passive avoidance was evaluated using water-finding and step-through tests, respectively.3. PrP ^–/– mice showed impaired performance in the water-finding test, indicating a disturbance in latent learning, at 23 weeks of age. In the step-through test, although the PrP ^–/– mice showed normal learning ability and short-term memory retention, they evidenced a significant disturbance in long-term memory retention.4. These results indicate that PrP^c is needed for certain types of learning and memory and that the loss of function of this protein may contribute to the pathogenesis of prion diseases.     

Convection-Enhanced Delivery of AAV2-PrPshRNA in Prion-Infected Mice

Prion disease is caused by a single pathogenic protein (PrP^Sc), an abnormal conformer of the normal cellular prion protein PrP^C. Depletion of PrP^C in prion knockout mice makes them resistant to prion disease. Thus, gene silencing of the Prnp gene is a promising effective therapeutic approach. Here, we examined adeno-associated virus vector type 2 encoding a short hairpin RNA targeting Prnp mRNA (AAV2-PrP-shRNA) to suppress PrP^C expression both in vitro and in vivo. AAV2-PrP-shRNA treatment suppressed PrP levels and prevented dendritic degeneration in RML-infected brain aggregate cultures. Infusion of AAV2-PrP-shRNA-eGFP into the thalamus of CD-1 mice showed that eGFP was transported to the cerebral cortex via anterograde transport and the overall PrP^C levels were reduced by ∼70% within 4 weeks. For therapeutic purposes, we treated RML-infected CD-1 mice with AAV2-PrP-shRNA beginning at 50 days post inoculation. Although AAV2-PrP-shRNA focally suppressed PrP^Sc formation in the thalamic infusion site by ∼75%, it did not suppress PrP^Sc formation efficiently in other regions of the brain. Survival of mice was not extended compared to the untreated controls. Global suppression of PrP^C in the brain is required for successful therapy of prion diseases.     

Mouse Prion Protein Polymorphism Phe-108/Val-189 Affects the Kinetics of Fibril Formation and the Response to Seeding: EVIDENCE FOR A TWO-STEP NUCLEATION POLYMERIZATION MECHANISM*

Prion diseases are fatal neurodegenerative disorders associated with the polymerization of the cellular form of prion protein (PrP^C) into an amyloidogenic β-sheet infectious form (PrP^Sc). The sequence of host PrP is the major determinant of host prion disease susceptibility. In mice, the presence of allele a (Prnp^a, encoding the polymorphism Leu-108/Thr-189) or b (Prnp^b, Phe-108/Val-189) is associated with short or long incubation times, respectively, following infection with PrP^Sc. The molecular bases linking PrP sequence, infection susceptibility, and convertibility of PrP^C into PrP^Sc remain unclear. Here we show that recombinant PrP^a and PrP^b aggregate and respond to seeding differently in vitro. Our kinetic studies reveal differences during the nucleation phase of the aggregation process, where PrP^b exhibits a longer lag phase that cannot be completely eliminated by seeding the reaction with preformed fibrils. Additionally, PrP^b is more prone to propagate features of the seeds, as demonstrated by conformational stability and electron microscopy studies of the formed fibrils. We propose a model of polymerization to explain how the polymorphisms at positions 108 and 189 produce the phenotypes seen in vivo. This model also provides insight into phenomena such as species barrier and prion strain generation, two phenomena also influenced by the primary structure of PrP.     

Shadoo/PrP (Sprn0/0/Prnp0/0) double knockout mice

Shadoo (Sho) is a brain glycoprotein with similarities to the unstructured region of PrP^C. Frameshift alleles of the Sho gene, Sprn, are reported in variant Creutzfeldt-Jakob disease (vCJD) patients while Sprn mRNA knockdown in PrP-null (Prnp^0/0) embryos produces lethality, advancing Sho as the hypothetical PrP-like “pi” protein. Also, Sho levels are reduced as misfolded PrP accumulates during prion infections. To penetrate these issues we created Sprn null alleles (Daude et al., Proc. Natl. Acad. Sci USA 2012; 109(23): 9035–40). Results from the challenge of Sprn null and TgSprn transgenic mice with rodent-adapted prions coalesce to define downregulation of Sho as a “tracer” for the formation of misfolded PrP. However, classical BSE and rodent-adapted BSE isolates may behave differently, as they do for other facets of the pathogenic process, and this intriguing variation warrants closer scrutiny. With regards to physiological function, double knockout mice (Sprn^0/0/Prnp^0/0) mice survived to over 600 d of age. This suggests that Sho is not pi, or, given the accumulating data for many activities for PrP^C, that the pi hypothesis invoking a discrete signaling pathway to maintain neuronal viability is no longer tenable.