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.     

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.     

Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

ABSTRACT

Prion diseases involve the conversion of the endogenous cellular prion protein, PrP^C, into a misfolded infectious isoform, PrP^Sc. Several functions have been attributed to PrP^C, and its role has also been investigated in the olfactory system. PrP^C is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp^?/? mice showed impaired behavior in olfactory tests. Given the high PrP^C expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrP^C-binding partners. Ten different putative PrP^C ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrP^C with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrP^C-Stub1 interaction are under investigation. The PrP^C-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrP^C is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein.     

Functions of the cellular prion protein,the end of Moore's law,and Ockham's razor theory

Since its discovery the cellular prion protein (encoded by the Prnp gene) has been associated with a large number of functions. The proposed functions rank from basic cellular processes such as cell cycle and survival to neural functions such as behavior and neuroprotection, following a pattern similar to that of Moore's law for electronics. In addition, particular interest is increasing in the participation of Prnp in neurodegeneration. However, in recent years a redefinition of these functions has begun, since examples of previously attributed functions were increasingly re-associated with other proteins. Most of these functions are linked to so-called “Prnp-flanking genes” that are close to the genomic locus of Prnp and which are present in the genome of some Prnp mouse models. In addition, their role in neuroprotection against convulsive insults has been confirmed in recent studies. Lastly, in recent years a large number of models indicating the participation of different domains of the protein in apoptosis have been uncovered. However, after more than 10 years of molecular dissection our view is that the simplest mechanistic model in PrP^C-mediated cell death should be considered, as Ockham's razor theory suggested.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Normal neurogenesis and scrapie pathogenesis in neural grafts lacking the prion protein homologue Doppel

The agent that causes prion diseases is thought to be identical to PrP^Sc, a conformer of the normal prion protein PrP^C. Recently a novel protein, termed Doppel (Dpl), was identified that shares significant biochemical and structural homology with PrP^C. To investigate the function of Dpl in neurogenesis and in prion pathology, we generated embryonic stem (ES) cells harbouring a homozygous disruption of the Prnd gene that encodes Dpl. After in vitro differentiation and grafting into adult brains of PrP^C-deficient Prnp^0/0 mice, Dpl-deficient ES cell-derived grafts contained all neural lineages analyzed, including neurons and astrocytes. When Prnd-deficient neural tissue was inoculated with scrapie prions, typical features of prion pathology including spongiosis, gliosis and PrP^Sc accumulation, were observed. Therefore, Dpl is unlikely to exert a cell-autonomous function during neural differentiation and, in contrast to its homologue PrP^C, is dispensable for prion disease progression and for generation of PrP^Sc.     

Mapping the Prion Protein Using Recombinant Antibodies

The fundamental event in prion disease is thought to be the posttranslational conversion of the cellular prion protein (PrP^C) into a pathogenic isoform (PrP^Sc). The occurrence of PrP^C on the cell surface and PrP^Sc in amyloid plaques in situ or in aggregates following purification complicates the study of the molecular events that underlie the disease process. Monoclonal antibodies are highly sensitive probes of protein conformation which can be used under these conditions. Here, we report the rescue of a diverse panel of 19 PrP-specific recombinant monoclonal antibodies from phage display libraries prepared from PrP deficient (Prnp^0/0) mice immunized with infectious prions either in the form of rods or PrP 27-30 dispersed into liposomes. The antibodies recognize a number of distinct linear and discontinuous epitopes that are presented to a varying degree on different PrP preparations. The epitope reactivity of the recombinant PrP(90-231) molecule was almost indistinguishable from that of PrP^C on the cell surface, validating the importance of detailed structural studies on the recombinant molecule. Only one epitope region at the C terminus of PrP was well presented on both PrP^C and PrP^Sc, while epitopes associated with most of the antibodies in the panel were present on PrP^C but absent from PrP^Sc.     

Involvement of Cellular Prion Protein in α-Synuclein Transport in Neurons

The cellular prion protein, encoded by the gene Prnp, has been reported to be a receptor of β-amyloid. Their interaction is mandatory for neurotoxic effects of β-amyloid oligomers. In this study, we aimed to explore whether the cellular prion protein participates in the spreading of α-synuclein. Results demonstrate that Prnp expression is not mandatory for α-synuclein spreading. However, although the pathological spreading of α-synuclein can take place in the absence of Prnp, α-synuclein expanded faster in PrP^C-overexpressing mice. In addition, α-synuclein binds strongly on PrP^C-expressing cells, suggesting a role in modulating the effect of α-synuclein fibrils.     

Dissecting the copper bioinorganic chemistry of the functional and pathological roles of the prion protein: Relevance in Alzheimer's disease and cancer

The cellular prion protein (PrP^C) is a metal-binding biomolecule that can interact with different protein partners involved in pivotal physiological processes, such as neurogenesis and neuronal plasticity. Recent studies profile copper and PrP^C as important players in the pathological mechanisms of Alzheimer's disease and cancer. Although the copper-PrP^C interaction has been characterized extensively, the role of the metal ion in the physiological and pathological roles of PrP^C has been barely explored. In this article, we discuss how copper binding and proteolytic processing may impact the ability of PrP^C to recruit protein partners for its functional roles. The importance to dissect the role of copper-PrP^C interactions in health and disease is also underscored.     

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.     

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.