Dose-dependent, prion protein (PrP)-mediated facilitation of excitatory synaptic transmission in the mouse hippocampus

Disruption of both alleles of the prion protein gene, Prnp, has been shown repeatedly to abolish the susceptibility of mice to developing prion diseases. However, conflicting results have been obtained from phenotypic analyses of prion protein (PrP)-deficient (Prnp0/0) mice lines. To explore the possible neurophysiological properties associated with expression or absence of the normal isoform of the cellular prion protein (PrPC), we used conventional in vitro extracellular field potential recordings in the hippocampal CA1 area of mice from two independently-derived Prnp0/0 strains. Basal synaptic transmission and a short-term form of synaptic plasticity were analysed in this study. Results were compared with animals carrying a wild-type mouse PrP transgene to investigate whether PrP expression levels influence glutamatergic synaptic transmission in the hippocampus. There was a clear correlation between excitatory synaptic transmission and PrP expression; i.e. the range of synaptic responses increased with the level of PrPC expression. On the other hand, the probability of transmitter release, as assessed by paired-pulse facilitation, appeared unchanged. Interestingly, whereas the overall range for synaptic responses was still greater in older mice over-expressing PrPC, this effect in these animals appeared to be due to better recruitment of fibres rather than facilitation of synaptic transmission per se. Taken together, these data are strong evidence for a functional role for PrPC in modulating synaptic transmission.     

Cell mediated immune responses against human prion protein

Vaccination approaches that may provide protection against the abnormal form of prion protein (PrPSc) have recently focused on the ability of antibodies to prevent PrPSc propagation. Progress has been hampered due to the difficulty in generating antibody responses in wild type mice, which is believed to be a consequence of T cell tolerance to the normal form of prion protein (PrPC). The problem of tolerance can be avoided using transgenic mice unable to express PrPC. This study examines active PrP specific T cell responses that can be produced in PrP null (PrP 0/0) mice using simple peptide vaccination procedures. Spleenocytes recovered from vaccinated PrP 0/0 mice were tested in vitro for their specificity with T cell recognition demonstrated through a proliferative response to the peptide. Analysis of mRNA also indicates the stimulation of a heterogenous population of T cells with an increase in cytokines and cytotoxicity associated mRNA. Responsive T cells were expanded using a T cell cloning procedure and demonstrated an ability to recognize the mature human prion protein. These clones may potentially be used to negate the problem of T cell tolerance in wild type mice.     

Packaging of prions into exosomes is associated with a novel pathway of PrP processing

Prion diseases are fatal, transmissible neurodegenerative disorders associated with conversion of the host-encoded prion protein (PrP(C)) into an abnormal pathogenic isoform (PrP(Sc)). Following exposure to the infectious agent (PrP(Sc)) in acquired disease, infection is propagated in lymphoid tissues prior to neuroinvasion and spread within the central nervous system. The mechanism of prion dissemination is perplexing due to the lack of plausible PrP(Sc)-containing mobile cells that could account for prion spread between infected and uninfected tissues. Evidence exists to demonstrate that the culture media of prion-infected neuronal cells contain PrP(Sc) and infectivity but the nature of the infectivity remains unknown. In this study we have identified PrP(C) and PrP(Sc) in association with endogenously expressing PrP neuronal cell-derived exosomes. The exosomes from our prion-infected neuronal cell line were efficient initiators of prion propagation in uninfected recipient cells and to non-neuronal cells. Moreover, our neuronal cell line was susceptible to infection by non-neuronal cell-derived exosome PrP(Sc). Importantly, these exosomes produced prion disease when inoculated into mice. Exosome-associated PrP is packaged via a novel processing pathway that involves the N-terminal modification of PrP and selection of distinct PrP glycoforms for incorporation into these vesicles. These data extend our understanding of the relationship between PrP and exosomes by showing that exosomes can establish infection in both neighbouring and distant cell types and highlight the potential contribution of differentially processed forms of PrP in disease distribution. These data suggest that exosomes represent a potent pool of prion infectivity and provide a mechanism for studying prion spread and PrP processing in cells endogenously expressing PrP.     

Establishment of the cell line expressing human prion protein on PrP0/0 background

In this work we described preparation of the novel cell lines expressing human prion protein on PrP0/0-background. Prepared cell lines originated from the Nagasaki mice (PrP0/0) and showed a fibroblast phenotype. The expression level of human prion protein in the developed cell lines was comparable to the physiological expression measured in GT1-7 cells. A great advantage of the prepared cell lines was their short doubling time that allowed obtaining of a large amount of cells for the proteomic experiments. Newly established cell lines open a broad spectrum of applications in the prion research. Besides the study of physiological function of the prion protein or its interactome, the new cell lines could be successfully employed as a unique tool for the better understanding of key events in the pathogenesis of prion diseases.     

Involvement of the cellular prion protein in the migration of brain microvascular endothelial cells

The conversion of cellular prion protein (PrP(C)) to its protease-resistant isoform is involved in the pathogenesis of prion disease. Although PrP(C) is a ubiquitous glycoprotein that is present in various cell types, the physiological role of PrP(C) remains obscure. The present study aimed to determine whether PrP(C) mediates migration of brain microvascular endothelial cells. Small interfering RNAs (siRNAs) targeting PrP(C) were transfected into a mouse brain microvascular endothelial cell line (bEND.3 cells). siPrP1, selected among three siRNAs, reduced mRNA and protein levels of PrP(C) in bEND.3 cells. Cellular migration was evaluated with a scratch-wound assay. siPrP1 suppressed migration without significantly affecting cellular proliferation. This study provides the first evidence that PrP(C) may be necessary for brain microvascular endothelial cells to migrate into damaged regions in the brain. This function of PrP(C) in the brain endothelium may be a mechanism by which the neurovascular unit recovers from an injury such as an ischemic insult.     

Overexpression of p62/SQSTM1 promotes the degradations of abnormally accumulated PrP mutants in cytoplasm and relieves the associated cytotoxicities via autophagy–lysosome-dependent way

The protein of p62/sequestosome 1 (SQSTM1), a key cargo adaptor protein involved in autophagy–lysosome degradation, exhibits inclusion bodies structure in cytoplasm and plays a protective role in some models of neurodegenerative diseases. Some PrP mutants, such as PrP-CYTO and PrP-PG14, also form cytosolic inclusion bodies and trigger neuronal apoptosis either in cultured cells or in transgenic mice. Here, we demonstrated that the cellular p62/SQSTM1 incorporated into the inclusion bodies formed by expressing the abnormal PrP mutants, PrP-CYTO and PrP-PG14, in human embryonic kidney 293 cells. Overexpression of p62/SQSTM1 efficiently relieved the cytosolic aggregations and cell apoptosis induced by the abnormal PrPs. Autophagy–lysosome inhibitors instead of proteasome inhibitor sufficiently blocked the p62/SQSTM1-mediated degradations of abnormal PrPs. Overexpression of p62/SQSTM1 did not alter the levels of light chain 3 (LC3) in the cells expressing various PrPs. However, more complexes of p62/SQSTM1 with LC3 were detected in the cells expressing the misfolded PrPs. These data imply that p62/SQSTM1 plays an important role in the homeostasis of abnormal PrPs via autophagy–lysosome-dependent way.     

A novel epitope for the specific detection of exogenous prion proteins in transgenic mice and transfected murine cell lines

Prion diseases are closely linked to the conversion of host-encoded cellular prion protein (PrPC) into its pathological isoform (PrPSc). PrP conversion experiments in scrapie infected tissue culture cells, transgenic mice, and cell-free systems usually require unique epitopes and corresponding monoclonal antibodies (MAbs) for the immunological discrimination of exogenously introduced and endogenous PrP compounds (e.g., MAb 3F4, which is directed to an epitope on hamster and human but not on murine PrP). In the current work, we characterize a novel MAb designated L42 that reacts to PrP of a variety of species, including cattle, sheep, goat, dog, human, cat, mink, rabbit, and guinea pig, but does not bind to mouse, hamster, and rat PrP. Therefore, MAb L42 may allow future in vitro conversion and transgenic studies on PrPs of the former species. The MAb L42 epitope on PrPC includes a tyrosine residue at position 144, whereas mouse, rat, and hamster PrPs incorporate tryptophane at this site. To verify this observation, we generated PrP expression vectors coding for authentic or mutated murine PrPCs (i.e., codon 144 encoding tyrosine instead of tryptophan). After transfection into neuroblastoma cells, MAb L42 did not react with immunoblotted wild-type murine PrPC, whereas L42 epitope-tagged murine PrPC was strongly recognized. Immunoblot and fluorescence-activated cell sorting data revealed that tagged PrPC was correctly posttranslationally processed and translocated to the cell surface.     

Close vicinity of PrP expressing cells (FDC) with noradrenergic fibers in healthy sheep spleen

In naturally and experimentally occurring scrapie in sheep, prions invade the immune system and replicate in lymphoid organs. Here we analysed immunohistochemically, in seven spleens of 6-month-old healthy sheep, the nature of the cells expressing prion protein (PrP) potentially supporting prion replication, as well as their relationship with autonomic innervation. PrP was identified using either RB1 rabbit antiserum or 4F2 monoclonal antibody directed against AA 108-123 portion of the bovine and AA 79-92 of human prion protein respectively. Using double labelling analysis, we demonstrated that PrPc is expressed by follicular dendritic cells using a specific monoclonal antibody (CNA42). We also showed the close vicinity of these PrP expressing cells with noradrenergic fibers, using a polyclonal tyrosine hydroxylase antibody. Our results may help the study of the cellular requirements for the possible neuroinvasion from the spleen.     

Translocation of cellular prion protein to non-lipid rafts protects human prion-mediated neuronal damage

Prions are the causative agents of transmissible spongiform encephalopathies, such as variant Creutzfeldt-Jakob disease in humans. Cellular prion proteins (PrPC) connect with cholesterol- and glycosphingolipid-rich lipid rafts through association of their glycosyl-phosphatidylinositol (GPI) anchor with saturated raft lipids and interaction of their N-terminal regions. Our previous study showed that cellular cholesterol enrichment prevented PrP(106-126)-induced neuronal death. We have now studied the influence of membrane cholesterol in PrP(106-126)-mediated neurotoxicity and identified membrane domains involved in this activity. We found that PrPC is normally distributed in lipid rafts, but high membrane cholesterol levels as a result of cholesterol treatment led to the translocation of PrPC from lipid rafts to non-lipid rafts. Moreover, cholesterol-mediated PrPC translocation protects PrP(106-126)-mediated apoptosis and p-38 activation and caspase-3 activation. In a mitochondrial functional assay including mitochondrial transmembrane potential, cholesterol treatment prevented the loss of mitochondrial potential, translocation of Bax and cytochrome c by prion protein fragment. Our results indicate that modulation of the PrPC location appears to protect against neuronal cell death caused by prion peptides. The results of this study suggest that regulation of membrane cholesterol affects the translocation of PrPC, which in turn regulates PrP(106-126)-induced mitochondrial dysfunction and neurotoxicity.     

PrP及其缺失突变体与GFAP体外相互作用的初步研究

目的研究PrP蛋白与GFAP蛋白是否发生分子间相互作用以及PrP蛋白多肽链中与GFAP蛋白相互作用的区域。方法制备仓鼠脑组织匀浆上清,通过原核表达系统以及体外翻译系统表达了全长的小鼠GFAP蛋白、人PrP蛋白以及各种缺失突变体,利用Pull-down及免疫共沉淀实验检测PrP与GFAP的分子间相互作用。结果不仅重组的GFAP与PrP发生分子间的相互作用,而且脑组织中的GFAP与PrPC及PrPSc也发生相互作用。PrP与GFAP蛋白相互作用的区域位于PrP的C端第91至231位氨基酸。结论PrP及其缺失突变体与GFAP在体外能够发生分子间的相互作用,提示GFAP可能参与朊蛋白的正常生理功能或者朊病毒病的病理过程。     

The toxicity of the PrP106-126 prion peptide on cultured photoreceptors correlates with the prion protein distribution in the mammalian and human retina

In patients affected by Creutzfeldt-Jakob disease and in animals affected by transmissible spongiform encephalopathies, retinal functions are altered, and major spongiform changes are observed in the outer plexiform layer where photoreceptors have their synaptic terminals. In the present study, the prion protein PrP(c) was found to form aggregates in rod photoreceptor terminals from both rat and human retina, whereas no labeling was observed in cone photoreceptors. Discrete staining was also detected in the inner plexiform layer where the prion protein was located at human amacrine cell synapses. In mixed porcine retinal cell cultures, the PrP106-126 prion peptide triggered a 61% rod photoreceptor cell loss by apoptosis as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling, whereas cone photoreceptors were not affected. Amacrine cells were also reduced by 47% in contrast to ganglion cells. Although this cell loss was associated with a 5.5-fold increase in microglial cells, the strict correlation between the PrP(c) prion protein expression and the peptide toxicity suggested that this toxicity did not rely on the release of a toxic compound by glial cells. These results provide new insights into the retinal pathophysiology of prion diseases and illustrate advantages of adult retinal cell cultures to investigate prion pathogenic mechanisms.     

Recent developments in prion immunotherapy

Antibody-based immunotherapy may represent a realistic approach against prion diseases, given that antibodies to the cellular prion protein PrPC have been shown to antagonize deposition of the disease-associated prion protein (termed PrPSc) in in vitro assays and in laboratory animals. However, induction of protective antiprion immune responses in wild-type animals is difficult because of host tolerance to the endogenous PrPC. Several studies indicate that it might be possible to overcome tolerance to PrPC and induce immune responses to bacterially expressed, recombinant PrP. However, it is much more difficult to induce antibodies capable of recognizing native cell-surface PrPC, and there is reason to believe that the latter immune responses correlate with anti-prion protection. The difficulties involved in eliciting development of such anti-native PrPC immune responses may be partly intrinsic to B cells and, in addition, may reside in peripheral T helper tolerance.     

PrP octarepeats region determined the interaction with caveolin-1 and phosphorylation of caveolin-1 and Fyn

Caveolin-1 is one of the major constituents of caveolae. Both Cav-1 and PrP are plasma membrane proteins, which show active capacities for molecular interactions with many other proteins or agents, including themselves. Using yeast two-hybrid system and immunoprecipitation, we reconfirmed the molecular interaction between human Cav-1 and PrP. With co-immunoprecipitation tests, PrP^C–Cav-1 and PrP^Sc–Cav-1 complexes were identified in the brain homogenates of normal and scrapie agent 263K-infected hamsters, respectively. Transient expression of wild-type PrP (PrP-PG5) in HEK293 cells did not change the situation of Cav-1 and subsequent signal transduction pathways, while cross-linking of the expressed PrP with specific antibody induced remarkable colocalization of PrP and Cav-1 on the plasma membrane and significant increases of phosphorylated Cav-1 and phosphorylated Fyn. With deleted and inserted PrP mutants within octarepeat region, we observed obvious octarepeat-associated phenomena, including lower binding capacity with Cav-1 in vitro, unable to co-localize with Cav-1 in the cells and to induce up-regulation of p-Cav-1 and p-Fyn when removal of octarepeats in the context of full-length PrP. Moreover, we found that treatment on HEK293 cells with fibrous form of recombinant PrP protein led to up-regulating the levels of p-Cav-1 and p-Fyn. Our data here provide strong evidence that octarepeats of PrP are critical for the interaction between PrP and Cav-1. Significant alterations in the cultured cells, either the distributions of PrP and Cav-1 morphologically or the up-regulations of p-Cav-1 and p-Fyn, induced by antibody-mediated cross-linking or fibrous forms of PrP may suggest a possible internalization process of PrP^Sc.     

Bee venom phospholipase A2 prevents prion peptide induced-cell death in neuronal cells

Bee venom phospholipase A2 (bvPLA2) is a prototypic group III enzyme which consists of unique N-terminal and C-terminal domains and a central secretory PLA2 (sPLA2) domain. This sPLA2 domain is highly homologous with human group III sPLA2. Current evidence suggests that group III sPLA2 may affect some neuronal functions, such as neuritogenesis, neurotransmitter release and neuronal survival. The prion diseases are neurodegenerative disorders characterized by the conversion of the normal cellular prion (PrPC) to the misfolded isoform scrapie prion protein (PrPSc). PrPSc accumulation in the central nervous system (CNS) leads to neurotoxicity by inhibition of the PI3K/AKT pathway or activation of p38 mitogen-activated protein kinase (MAPK) pathways. In the present study, we found that bvPLA2 inhibited prion protein (PrP) fragment (106-126)-induced neuronal cell death. PrP(106-126)-mediated increase of p-p38 MAPK and cleaved caspases and decrease of p-AKT were blocked by bvPLA2 treatment. These results indicate that increasing PLA2, including the group III sPLA2 is key to regulating PrP(106-126)-mediated neurotoxicity. Taken together, the results of this study suggest that specific modulation of PLA2 appears to prevent neuronal cell death caused by prion peptides.     

Mitochondrial localization of cellular prion protein (PrPC) invokes neuronal apoptosis in aged transgenic mice overexpressing PrPC

Recent studies suggest that the disease isoform of prion protein (PrPSc) is non-neurotoxic in the absence of cellular isoform of prion protein (PrPC), indicating that PrPC may participate directly in the neurodegenerative damage by itself. Meanwhile, transgenic mice harboring a high-copy-number of wild-type mouse (Mo) PrPC develop a spontaneous neurological dysfunction in an age-dependent manner, even without inoculation of PrPSc and thus, investigations of these aged transgenic mice may lead to the understanding how PrPC participate in the neurotoxic property of PrP. Here we demonstrate mitochondria-mediated neuronal apoptosis in aged transgenic mice overexpressing wild-type MoPrPC (Tg(MoPrP)4053/FVB). The aged mice exhibited an aberrant mitochondrial localization of PrPC concomitant with decreased proteasomal activity, while younger littermates did not. Such aberrant mitochondrial localization was accompanied by decreased mitochondrial manganese superoxide dismutase (Mn-SOD) activity, cytochrome c release into the cytosol, caspase-3 activation, and DNA fragmentation, most predominantly in hippocampal neuronal cells. Following cell culture studies confirmed that decrease in the proteasomal activity is fundamental for the PrPC-related, mitochondria-mediated apoptosis. Hence, the neurotoxic property of PrPC could be explained by the mitochondria-mediated neuronal apoptosis, at least in part.     

Hypoxia-inducible factor-1 alpha regulates prion protein expression to protect against neuron cell damage

The human prion protein fragment, PrP (106-126), may contain a majority of the pathological features associated with the infectious scrapie isoform of PrP, known as PrP(Sc). Based on our previous findings that hypoxia protects neuronal cells from PrP (106-126)-induced apoptosis and increases cellular prion protein (PrP(C)) expression, we hypothesized that hypoxia-related genes, including hypoxia-inducible factor-1 alpha (HIF-1α), may regulate PrP(C) expression and that these genes may be involved in prion-related neurodegenerative diseases. Hypoxic conditions are known to elicit cellular responses designed to improve cell survival through adaptive processes. Under normoxic conditions, a deferoxamine-mediated elevation of HIF-1α produced the same effect as hypoxia-inhibited neuron cell death. However, under hypoxic conditions, doxorubicin-suppressed HIF-1α attenuated the inhibitory effect on neuron cell death mediated by PrP (106-126). Knock-down of HIF-1α using lentiviral short hairpin (sh) RNA-induced downregulation of PrP(C) mRNA and protein expression under hypoxic conditions, and sensitized neuron cells to prion peptide-mediated cell death even in hypoxic conditions. In PrP(C) knockout hippocampal neuron cells, hypoxia increased the HIF-1α protein but the cells did not display the inhibitory effect of prion peptide-induced neuron cell death. Adenoviruses expressing the full length Prnp gene (Ad-Prnp) were utilized for overexpression of the Prnp gene in PrP(C) knockout hippocampal neuron cells. Adenoviral transfection of PrP(C) knockout cells with Prnp resulted in the inhibition of prion peptide-mediated cell death in these cells. This is the first report demonstrating that expression of normal PrP(C) is regulated by HIF-1α, and PrP(C) overexpression induced by hypoxia plays a pivotal role in hypoxic inhibition of prion peptide-induced neuron cell death. These results suggest that hypoxia-related genes, including HIF-1α, may be involved in the pathogenesis of prion-related diseases and as such may be a therapeutic target for prion-related neurodegenerative diseases.     

Prion Protein Transgenes and the Neuropathology in Prion Diseases

The concept that prions are novel pathogens which are different from both viroids and viruses has received increasing support from many avenues of investigation over the past decade. Enriching fractions from Syrian hamster (SHa) brain for scrapie prion infectivity led to the discovery of the prion protein (PrP). Prion diseases of animals include scrapie and “mad cow” disease; those of humans present as inherited, sporadic and infectious neurodegenerative disorders, two of which are called Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker disease (GSS). The inherited human prion diseases are genetically linked to mutations in the PrP gene that result in non-conservative amino acid substitutions. Transgenic (Tg) mice expressing PrP carrying a GSS mutation developed neurodegeneration spontaneously and produced prions de novo. In other studies, Tg mice expressing both SHa and mouse (Mo) PrP genes were used to demonstrate that the “species barrier” for scrapie prions resides in the primary structure of PrP. This concept was strengthened by the results of studies in which mice expressing chimeric Mo/human (Hu) PrP transgenes were constructed which differ from MoPrP by nine amino acids between residues 96 and 167. All of the Tg(MHu2M) mice developed neurologic disease ～200 days after inoculation with brain homogenate from three patients who died of CJD. About 10% of Tg(HuPrP) mice expressing HuPrP and non-Tg mice developed neurologic disease >500 days after inoculation with CJD prions. The different susceptibilities of Tg(HuPrP) and Tg(MHu2M) mice to human prions indicate that additional species specific factors such as chaperone proteins are involved in prion replication. Diagnosis, prevention and treatment of human prion diseases should be facilitated by study of Tg(MHu2M) mice. Our findings and those from other studies suggest that mutant and wtPrP interact, perhaps through a chaperone-like protein, during the pathogenesis of the prion diseases.     

Differential inhibition of prion propagation by enantiomers of quinacrine

Prion diseases are fatal neurologic disorders caused by accumulation of a pathogenic isoform (PrP(Sc)) of the prion protein (PrP). The recent discovery of the inhibitory action of quinacrine on PrP(Sc) formation in scrapie-infected neuroblastoma (ScN2a) cells raised the possibility of a treatment for patients with prion disease. To investigate the efficacy of quinacrine enantiomers, we measured the inhibitory effect of these isomers on PrP(Sc) formation in ScN2a cells. (S)-quinacrine exhibited superior antiprion activity compared with (R)-quinacrine and two generic quinacrines that appear to be racemates. Treatment with these various forms of quinacrine did not induce adverse changes affecting cell survival and the expression of marker proteins over a range of potentially therapeutic concentrations. Thus, quinacrine enantiomers demonstrated stereoselectivity on prion elimination but not cytotoxicity in ScN2a cells. Our results raise the possibility that in vivo treatment using one enantiomer of quinacrine may be superior to a racemic mixture, which is the form that is generally used when quinacrine is employed to treat parasitic diseases.