金黄地鼠PrP基因组织特异性表达的研究

金黄地鼠是研究动物传染性海绵状脑病的理想模型动物之一,我们利用实时荧光定量RT-PCR技术,构建标准重组质粒制备标准曲线,对中枢神经系统的4个不同部位及外周6个组织提取总RNA,反转录后进行PrP基因表达的定量.结果发现,脑的四个检测部位都呈现高的表达量;在外周器官中,淋巴结的表达量和全脑相当,脾脏、心脏、肝脏和肺脏呈现中等程度的表达,肾脏的表达量最低.本研究的结果对于探讨朊蛋白的基本功能和不同组织在传染性海绵状病理发生中的作用,提供了基础数据.     

Cre-loxP mediated control of PrP to study transmissible spongiform encephalopathy diseases

Expression of the PrP glycoprotein is essential for the development of the transmissible spongiform encephalopathy (TSE) or prion diseases. Although PrP is widely expressed in the mouse, the precise relevance of different PrP-expressing cell types to disease remains unclear. To address this, we generated two lines of floxed PrP gene-targeted transgenic mice using the Cre recombinase-loxP system. These floxed mice allow a functional PrP allele to be either switched "on" or "off." We demonstrate control of PrP expression for both alleles following Cre-mediated recombination, as determined by PrP mRNA and protein expression in the brain. Moreover, we show that Cre-mediated alteration of PrP expression in these mice has a major influence on the development of TSE disease. These floxed PrP mice will allow the involvement of PrP expression in specific cell types following TSE infection to be defined, which may identify potential sites for therapeutic intervention.     

Adaptation and selection of prion protein strain conformations following interspecies transmission of transmissible mink encephalopathy

Interspecies transmission of the transmissible spongiform encephalopathies (TSEs), or prion diseases, can result in the adaptation and selection of TSE strains with an expanded host range and increased virulence such as in the case of bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease. To investigate TSE strain adaptation, we serially passaged a biological clone of transmissible mink encephalopathy (TME) into Syrian golden hamsters and examined the selection of distinct strain phenotypes and conformations of the disease-specific isoform of the prion protein (PrP(Sc)). The long-incubation-period drowsy (DY) TME strain was the predominate strain, based on the presence of its strain-specific PrP(Sc) following interspecies passage. Additional serial passages in hamsters resulted in the selection of the hyper (HY) TME PrP(Sc) strain-dependent conformation and its short incubation period phenotype unless the passages were performed with a low-dose inoculum (e.g., 10(-5) dilution), in which case the DY TME clinical phenotype continued to predominate. For both TME strains, the PrP(Sc) strain pattern preceded stabilization of the TME strain phenotype. These findings demonstrate that interspecies transmission of a single cloned TSE strain resulted in adaptation of at least two strain-associated PrP(Sc) conformations that underwent selection until one type of PrP(Sc) conformation and strain phenotype became predominant. To examine TME strain selection in the absence of host adaptation, hamsters were coinfected with hamster-adapted HY and DY TME. DY TME was able to interfere with the selection of the short-incubation HY TME phenotype. Coinfection could result in the DY TME phenotype and PrP(Sc) conformation on first passage, but on subsequent passages, the disease pattern converted to HY TME. These findings indicate that during TSE strain adaptation, there is selection of a strain-specific PrP(Sc) conformation that can determine the TSE strain phenotype.     

PrPTSE distribution in a primate model of variant, sporadic, and iatrogenic Creutzfeldt-Jakob disease

Human prion diseases, such as Creutzfeldt-Jakob disease (CJD), are neurodegenerative and fatal. Sporadic CJD (sCJD) can be transmitted between humans through medical procedures involving highly infected organs, such as the central nervous system. However, in variant CJD (vCJD), which is due to human contamination with the bovine spongiform encephalopathy (BSE) agent, lymphoreticular tissue also harbors the transmissible spongiform encephalopathy-associated prion protein (PrP(TSE)), which poses a particularly acute risk for iatrogenic transmission. Two blood transfusion-related cases are already documented. In addition, the recent observation of PrP(TSE) in spleen and muscle in sCJD raised the possibility that peripheral PrP(TSE) is not limited to vCJD cases. We aimed to clarify the peripheral pathogenesis of human TSEs by using a nonhuman primate model which mimics human diseases. A highly sensitive enzyme-linked immunosorbent assay was adapted to the detection of extraneural PrP(TSE). We show that affected organs can be divided into two groups. The first is peripheral organs accumulating large amounts of PrP(TSE), which represent a high risk of iatrogenic transmission. This category comprises only lymphoreticular organs in the vCJD/BSE model. The second is organs with small amounts of PrP(TSE) associated with nervous structures. These are the muscles, adrenal glands, and enteric nervous system in the sporadic, iatrogenic, and variant CJD models. In contrast to the first set of organs, this low level of tissue contamination is not strain restricted and seems to be linked to secondary centrifugal spread of the agent through nerves. It might represent a risk for iatrogenic transmission, formerly underestimated despite previous reports of low rates of transmission from peripheral organs of humans to nonhuman primates (5, 10). This study provides an additional experimental basis for the classification of human organs into different risk categories and a rational re-evaluation of current risk management measures.     

Transmission of chronic wasting disease identifies a prion strain causing cachexia and heart infection in hamsters

Chronic wasting disease (CWD) is an emerging prion disease of free-ranging and captive cervids in North America. In this study we established a rodent model for CWD in Syrian golden hamsters that resemble key features of the disease in cervids including cachexia and infection of cardiac muscle. Following one to three serial passages of CWD from white-tailed deer into transgenic mice expressing the hamster prion protein gene, CWD was subsequently passaged into Syrian golden hamsters. In one passage line there were preclinical changes in locomotor activity and a loss of body mass prior to onset of subtle neurological symptoms around 340 days. The clinical symptoms included a prominent wasting disease, similar to cachexia, with a prolonged duration. Other features of CWD in hamsters that were similar to cervid CWD included the brain distribution of the disease-specific isoform of the prion protein, PrP(Sc), prion infection of the central and peripheral neuroendocrine system, and PrP(Sc) deposition in cardiac muscle. There was also prominent PrP(Sc) deposition in the nasal mucosa on the edge of the olfactory sensory epithelium with the lumen of the nasal airway that could have implications for CWD shedding into nasal secretions and disease transmission. Since the mechanism of wasting disease in prion diseases is unknown this hamster CWD model could provide a means to investigate the physiological basis of cachexia, which we propose is due to a prion-induced endocrinopathy. This prion disease phenotype has not been described in hamsters and we designate it as the 'wasting' or WST strain of hamster CWD.     

Host PrP glycosylation: a major factor determining the outcome of prion infection

The expression of the prion protein (PrP) is essential for transmissible spongiform encephalopathy (TSE) or prion diseases to occur, but the underlying mechanism of infection remains unresolved. To address the hypothesis that glycosylation of host PrP is a major factor influencing TSE infection, we have inoculated gene-targeted transgenic mice that have restricted N-linked glycosylation of PrP with three TSE strains. We have uniquely demonstrated that mice expressing only unglycosylated PrP can sustain a TSE infection, despite altered cellular location of the host PrP. Moreover we have shown that brain material from mice infected with TSE that have only unglycosylated PrP^Sc is capable of transmitting infection to wild-type mice, demonstrating that glycosylation of PrP is not essential for establishing infection within a host or for transmitting TSE infectivity to a new host. We have further dissected the requirement of each glycosylation site and have shown that different TSE strains have dramatically different requirements for each of the glycosylation sites of host PrP, and moreover, we have shown that the host PrP has a major role in determining the glycosylation state of de novo generated PrP^Sc.     

Degradation of the disease-associated prion protein by a serine protease from lichens

The disease-associated prion protein (PrP(TSE)), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrP(TSE) inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrP(TSE). Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrP(TSE)-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrP(TSE) and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.     

Entry versus blockade of brain infection following oral or intraperitoneal scrapie administration: role of prion protein expression in peripheral nerves and spleen

Naturally occurring transmissible spongiform encephalopathy (TSE) diseases such as bovine spongiform encephalopathy in cattle are probably transmitted by oral or other peripheral routes of infection. While prion protein (PrP) is required for susceptibility, the mechanism of spread of infection to the brain is not clear. Two prominent possibilities include hematogenous spread by leukocytes and neural spread by axonal transport. In the present experiments, following oral or intraperitoneal infection of transgenic mice with hamster scrapie strain 263K, hamster PrP expression in peripheral nerves was sufficient for successful infection of the brain, and cells of the spleen were not required either as a site of amplification or as transporters of infectivity. The role of tissue-specific PrP expression of foreign PrP in interference with scrapie infection was also studied in these transgenic mice. Peripheral expression of heterologous PrP completely protected the majority of mice from clinical disease after oral or intraperitoneal scrapie infection. Such extensive protection has not been seen in earlier studies on interference, and these results suggested that gene therapy with mutant PrP may be effective in preventing TSE diseases.     

PrP 106-126 Altered PrP mRNA gene expression in mouse microglia BV-2 Cells

Prion diseases are infectious and fatal neurodegenerative diseases. The pathogenic agent is an abnormal prion protein aggregate. Microglial activation in the centre nervous system is a characteristic feature of prion disease. In this study, we examined the effect of PrP 106–126 on PrP mRNA gene expression in Mouse microglia cells BV-2 by real-time quantitative PCR. PrP mRNA expression level was found to be significantly increased after 18 h exposure of BV-2 cells to PrP 106–126, with 3-fold increase after 18 h and 4.5-fold increase after 24 h and BV-2 cells proliferating occurred correspondingly. Our results provide the first in vitro evidence of the increase of PrP mRNA levels in microglial cells exposed to PrP 106–126, and indicate that microglial cells might play a critical role in prion pathogenesis.     

Quantitative profiling of the pathological prion protein allotypes in bank voles by liquid chromatography-mass spectrometry

The conversion of the cellular prion protein (PrP(C)) into a misfolded isoform (PrP(TSE)) that accumulates in the brain of affected individuals is the key feature of transmissible spongiform encephalopaties (TSEs). Susceptibility to TSEs is influenced by polymorphisms of the prion gene suggesting that the presence of certain amino acid residues may facilitate the pathological conversion. In this work, we describe a quantitative, fast and reliable HPLC-MS method that allowed to demonstrate that in the brain of 109(Met/Ile) heterozygous bank voles infected with the mouse adapted scrapie strain 139A, there are comparable amounts of PrP(TSE) with methionine or isoleucine in position 109, suggesting that in this TSE model the two allotypes have similar rates of accumulation. This method can be easily adapted for the quantitative determination of PrP allotypes in the brain of other natural or experimental TSE models.     

Correlation between Infectivity and Disease Associated Prion Protein in the Nervous System and Selected Edible Tissues of Naturally Affected Scrapie Sheep

The transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative disorders characterised by the accumulation of a pathological form of a host protein known as prion protein (PrP). The validation of abnormal PrP detection techniques is fundamental to allow the use of high-throughput laboratory based tests, avoiding the limitations of bioassays. We used scrapie, a prototype TSE, to examine the relationship between infectivity and laboratory based diagnostic tools. The data may help to optimise strategies to prevent exposure of humans to small ruminant TSE material via the food chain. Abnormal PrP distribution/accumulation was assessed by immunohistochemistry (IHC), Western blot (WB) and ELISA in samples from four animals. In addition, infectivity was detected using a sensitive bank vole bioassay with selected samples from two of the four sheep and protein misfolding cyclic amplification using bank vole brain as substrate (vPMCA) was also carried out in selected samples from one animal. Lymph nodes, oculomotor muscles, sciatic nerve and kidney were positive by IHC, WB and ELISA, although at levels 100–1000 fold lower than the brain, and contained detectable infectivity by bioassay. Tissues not infectious by bioassay were also negative by all laboratory tests including PMCA. Although discrepancies were observed in tissues with very low levels of abnormal PrP, there was an overall good correlation between IHC, WB, ELISA and bioassay results. Most importantly, there was a good correlation between the detection of abnormal PrP in tissues using laboratory tests and the levels of infectivity even when the titre was low. These findings provide useful information for risk modellers and represent a first step toward the validation of laboratory tests used to quantify prion infectivity, which would greatly aid TSE risk assessment policies.     

RNA aptamers specifically interact with the prion protein PrP.

We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein rPrP23-231 (rPrPc) fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST::rPrP90-231 (rPrP27-30), which lacks 67 amino acids from the PrP N terminus. The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition. Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice (C57BL/6), hamsters (Syrian golden), and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice (prn-p(0/0)). Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize PrP27-30 in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.     

多肽PrP106—126对培养神经细胞朊蛋白基因表达的影响

神经细胞是传染性海绵状脑病（transmissible spongiform encephalopathies,TSEs）的重要靶细胞,PrP106-126是研究TSEs致病机理的理想工具,对PrP106-126作用的培养神经细胞模型进行研究,有利于了解朊蛋白的功能和探讨TSEs的分子致病机制。本研究利用PrP106-126构建了大脑皮质和小脑颗粒神经元作用模型,对神经细胞的存活和朊蛋白基因的表达进行了研究。结果表明：PrP106-126作用于培养神经细胞导致其存活率的显著下降;大脑皮质神经元经PrP106-126处理后,与SCR处理组和对照组相比,基因表达的量明显下降,处理后的小脑颗粒神经元也有类似的情况出现,两者之间下降的幅度和时间不同。我们的研究结果为研究朊蛋白在TSEs发生中的作用和深入了解TSE的分子致病机制提供了基础数据。     

Follicular dendritic cell of the knock-in mouse provides a new bioassay for human prions

Infectious prion diseases initiate infection within lymphoid organs where prion infectivity accumulates during the early stages of peripheral infection. In a mouse-adapted prion infection, an abnormal isoform (PrP(Sc)) of prion protein (PrP) accumulates in follicular dendritic cells within lymphoid organs. Human prions, however, did not cause an accumulation of PrP(Sc) in the wild type mice. Here, we report that knock-in mouse expressing humanized chimeric PrP demonstrated PrP(Sc) accumulations in follicular dendritic cells following human prion infections, including variant Creutzfeldt-Jakob disease. The accumulated PrP(Sc) consisted of recombinant PrP, but not of the inoculated human PrP. These accumulations were detectable in the spleens of all mice examined 30 days post-inoculation. Infectivity of the spleen was also evident. Conversion of humanized PrP in the spleen provides a rapid and sensitive bioassay method to uncover the infectivity of human prions. This model should facilitate the prevention of infectious prion diseases.     

Nervous and nonnervous cell transduction by recombinant adenoviruses that inducibly express the human PrP.

The study of the prion protein (PrP) physiological functions or its specific role in transmissible spongiform encephalopathies (TSE) requires new tools, particularly those able to induce PrP overexpression in a large range of cells, in vivo as well as in vitro. Here we describe the construction of two recombinant adenoviruses encoding the human PrP either with a valine at position 129 (AdTRVal) or a methionine (AdTRMet). Both genes were put under the control of the tetracycline-responsive promoter, allowing tight regulation of PrP expression. AdTRVal and AdTRMet induced high expression of the human PrP in CHO-KI cells and in organotypic brain slices in culture. The proteins expressed from these viruses exhibited a glycosylphosphatidyl inositol (GPI) anchor, proper glycosylation and sensitivity to proteinase K digestion. AdTRVal and AdTRMet will allow future studies on the human PrP and on the role of the codon 129 polyphormism in human TSE.     

Prion protein gene expression in cultured cells

A single copy gene encodes both the scrapie (PrPSc) and cellular (PrPC) isoforms of the prion protein (PrP). Cultured cell lines were found to express the endogenous PrP mRNA at levels comparable to those observed in the brains of adult rodents; however, these cells were invariably found to express greatly reduced levels of PrP. In all the cell lines examined, PrP was undetectable by Western immunoblot analysis. These cells were also poor recipients for expression constructs linking the hamster PrP gene open reading frame to several strong eukaryotic promoters; stable clones derived by transfection of these expression vectors failed to show elevated expression of PrP. When extremely high levels of PrP mRNA were produced using either an insect baculovirus or a mammalian SV40 based vector, significant quantities of PrP were produced, although in both cases the proteins were apparently processed differently from the PrPC observed in brains. In an expression system using an SV40 late promoter vector in monkey COS-7 cells, a significant fraction of PrP was transported to the cell surface where PrPC is found in vivo. PrP synthesized by the baculovirus vector failed to induce scrapie in hamsters and did not possess the characteristics of the PrPSc isoform associated with infectivity. The SV40 late promoter vector system may permit experiments designed to elucidate the role of PrPSc during scrapie infection as well as the function of PrPC in normal metabolism.     

Remarkable increases of α1-antichymotrypsin in brain tissues of rodents during prion infection

α1-Antichymotrypsin (α1-ACT) belongs to a kind of acute-phase inflammatory protein. Recently, such protein has been proved exist in the amyloid deposits which is the hallmark of Alzheimer's disease, but limitedly reported in prion disease. To estimate the change of α1-ACT during prion infection, the levels of α1-ACT in the brain tissues of scrapie agents 263K-, 139A- and ME7-infected rodents were analyzed, respectively. Results shown that α1-ACT levels were significantly increased in the brain tissues of the three kinds of scrapie-infected rodents, displaying a time-dependent manner during prion infection. Immunohistochemistry assays revealed the increased α1-ACT mainly accumulated in some cerebral regions of rodents infected with prion, such as cortex, thalamus and cerebellum. Immunofluorescent assays illustrated ubiquitously localization of α1-ACT with GFAP positive astrocytes, Iba1-positive microglia and NeuN-positive neurons. Moreover, double-stained immunofluorescent assays and immunohistochemistry assays using series of brain slices demonstrated close morphological colocalization of α1-ACT signals with that of PrP and PrP^Sc in the brain slices of 263K-infected hamster. However, co-immunoprecipitation does not identify any detectable molecular interaction between the endogenous α1-ACT and PrP either in the brain homogenates of 263K-infected hamsters or in the lysates of prion-infected cultured cells. Our data here imply that brain α1-ACT is increased abnormally in various scrapie-infected rodent models. Direct molecular interaction between α1-ACT and PrP seems not to be essential for the morphological colocalization of those two proteins in the brain tissues of prion infection.     

Structural differences between TSEs strains investigated by FT-IR spectroscopy

Strain diversity in transmissible spongiform encephalopathies (TSEs) has been suggested to be "enciphered" in the structure of the misfolded prion protein isoform PrP(Sc). We have recently demonstrated the strain typing potential of the FT-IR spectroscopy technique, analyzing four different TSE agents adapted to Syrian hamsters [A. Thomzig, S. Spassov, M. Friedrich, D. Naumann and M. Beekes, Discriminating scrapie and BSE isolates by infrared spectroscopy of pathological prion protein J. Biol. Chem. 279 (2004) 33847-33854.] [1]. In the present paper, we have extended the FT-IR study, exploring the secondary structure, temperature stability, and hydrogen-deuterium exchange characteristics of PrP27-30, from the TSE agents 263K, ME7-H, 22A-H, and BSE-H. The strain differentiation capacity of the FT-IR approach was objectively proven for the first time by multivariate cluster analysis. The second derivative FT-IR spectra obtained from dried protein films or samples hydrated in H(2)O or D(2)O consistently exhibited strain-specific infrared characteristics in the secondary structure sensitive amide I region, complemented by strain dependent spectral traits in the amide II and amide A absorption regions, and the different H/D-exchange behaviour of the various PrP27-30 samples. FT-IR spectra of PrP27-30 samples from 263K, ME7-H and 22A-H exposed to increasing temperature (up to 90 degrees C) showed that a strain-specific response to heat treatment is associated with strain specific thermostability of distinct secondary structure elements, providing additional means for TSEs strain discrimination.     

Developmental expression of the cellular prion protein (PrP(C) ) in bovine embryos

The mammalian cellular prion protein (PrP(C) ) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrP(Sc) ), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about PrP(Sc) conversion and its role in TSEs, the normal function of PrP(C) has not been elucidated. In adult mammals, PrP(C) is most abundant in the central nervous tissue, with intermediate levels in the intestine and heart, and lower levels in the pancreas and liver. PrP(C) is expressed during neurogenesis throughout development, and it has recently been proposed that PrP(C) participates in neural cell differentiation during embryogenesis. In order to establish the developmental timing and to address the cell-specific expression of PrP(C) during mammalian development, we examined PrP(C) expression in bovine gametes and embryos through gestation Day 39. Our data revealed differential levels of Prnp mRNA at Days 4 and 18 in pre-attachment embryos. PrP(C) was detected in the developing central and peripheral nervous systems in Day-27, 32-, and -39 embryos. PrP(C) was particularly expressed in differentiated neural cells located in the marginal regions of the central nervous system, but was absent from mitotically active, periventricular areas. Moreover, a PrP(C) cell-specific pattern of expression was detected in non-nervous tissues, including liver and mesonephros, during these stages. The potential participation of PrP(C) in neural cell differentiation is supported by its specific expression in differentiated states of neurogenesis.