羊瘙羊因子263K毒株感染金黄地鼠后鼠脑组织中检出PrP—res蛋白及其神经病理学研究

羊瘙痒病是累及山羊及绵羊的传播海绵状脑病。为了观察羊瘙痒因子（Scrapie）的病原特征及病理组织改变特点,将羊瘙痒因子263K毒株颅内接种至金黄地鼠。经过81－110天的潜伏期,89％的动物发病（17／19只）。对发病地鼠的神经病理学检测发现海绵状空泡变性的检出率为59％,淀粉斑的检出率为17.6％,利用免疫组化和蛋白酶消化后Western blotting检测证实,100％的发病地鼠的脑组织中都出现蛋白酶抗性朊蛋白（PrP-res）。17只发病地鼠脑组织提取物中,PrP-res的泳动位置和分子量大小完全一致,出现两条分子量在25kD-31kD的反应带。尝试应用快速玻片印迹法检测病变组织中的PrP-res,结果显示,与常规因定包埋切片的免疫组化检出效果相似。这提示脑组织印片法可成为临床检测克－雅氏病（Creutzfeldt-Jacob disease,CJD)患者脑组织活检标本中PrP-res的快速、有效的方法,羊瘙痒因子263K成功感染金黄地鼠再次证明,金黄地鼠是TSE感染因子良好的动物模型,发病率高,潜伏期短,发病动物PrP-res的检出率明显高于典型病理改变的检出率,新生成的PrP-res的电泳类型与接种的TSE因子有关,与宿主的个体差异无关,提示TSE感染因子的确存在“株”的现象。     

羊瘙痒病263K毒株感染的仓鼠脑中PrP分子形式多样性的动态测定

正常细胞的朊蛋白(PrPC)代谢和构象的改变是引发动物和人类可传播性海绵状脑病(transmissiblespongiformencephalopathies,TSEs)的根本原因。将羊瘙痒病(scrapie)仓鼠适应株263K颅内接种仓鼠,在接种后的第20、40、50、60、70、80天,通过Westernblot动态检测仓鼠脑中PrP存在的形式。结果在接种后第40天,在感染动物脑组织中即检测到PrPSc分子,比临床症状出现的时间早(平均潜伏期为66 7±1 1天),且无糖基化形式的PrP分子所占百分比在接种后期增加明显。除了标准分子量大小(30kD～35kD)的PrP分子外,在感染动物脑中存在着高分子量和低分子量形式的PrP分子。定量分析显示,随着接种潜伏期的延长,不同形式PrP分子的含量也在增加,其中低分子量形式的PrP分子与临床症状的出现密切相关。蛋白去糖基化实验表明,在感染动物脑组织中,除了标准分子量大小的PrP蛋白外,还存在一条更小分子量的PrP条带,而正常动物脑组织仅存在标准大小的PrP分子。低分子量形式的PrP分子具有与全长PrP分子相类似的糖基化模式。结果提示,scrapie263K感染的仓鼠脑组织中存在不同分子形式的PrPSc,其PrP分子的代谢可能不同于正常动物。     

羊瘙痒病小鼠适应株139A可突破种属屏障颅内感染金黄地鼠

羊瘙痒病感染因子可以根据疾病发生的潜伏期、临床病程、神经病理学特征以及PrPSc分子特征等分为不同的毒株,目前已经证实有20余种.在可传播性海绵状脑病的发病过程中存在着明显的种属屏障作用.将小鼠适应株139A颅内接种至金黄地鼠,以观测感染因子对种属屏障的突破及感染特征.在接种385～405天后,感染动物出现明显症状,与以往报道的金黄地鼠适应株263K不同,139A毒株发病动物出现严重的瘙痒,而无明显的共济失调.感染动物自出现明显症状到死亡的时间明显长于263K毒株感染金黄地鼠.进一步脑组织电镜和Western blot检测证实,存在有羊瘙痒病相关纤维和PrP-res.这证明小鼠适应株139A可突破种属屏障感染金黄地鼠.经系统比较,139A和263K毒株在潜伏期、主要临床症状和临床病程显示出明显的差异,而PrP-res的泳动位置和糖基化比率差异不大,仅139A毒株的单糖基化分子位置似乎略高于263K.动态观测处于潜伏期的接种动物脑组织羊瘙痒病相关纤维和PrP-res,发现PrPSc的出现明显早于临床症状.     

免疫印迹法检测牛海绵状脑病和羊瘙痒病

用大肠杆菌表达的牛朊病毒正常成熟蛋白 (BoPrPC)免疫新西兰白兔 ,获得了与朊病毒蛋白 (PrP)反应的抗体T1。根据致病型朊病毒 (PrP^SC)能抵抗蛋白酶消化的特性 ,用蛋白酶K消化脑组织提取物 ,以抗体T1进行免疫印迹反应 ,结果表明从接种羊瘙痒病朊病毒 2 6 3K的金黄地鼠脑组织提取物内检测到抗蛋白酶K消化的致病型PrPSC ,而正常金黄地鼠脑组织中没有抗蛋白酶消化的蛋白。以我国正常牛羊为材料 ,制备其脑组织提取物 ,用上述方法和抗体T1进行检测 ,结果没有发现抗蛋白酶K的任何蛋白存在 ,说明没有牛海绵状脑病和羊瘙痒病存在。用 1A8抗体也获得了同样的结果。这些结果表明可以用自制的抗血清检疫牛海绵状脑病和羊瘙痒病 ,防止其传入我国     

一种新的PrP无细胞构象转化系统的建立

PrP细胞外构象转化系统已经被一些实验室用于朊病毒的研究,但在反应体系中往往需要使用放射性同位素。为了建立一种安全方便的PrP无细胞构象转化系统用于TSE发病机制的研究,通过PCR方法获得仓鼠PrP(HaPrP)全长基因,克隆至原核表达载体pQE30,在大肠杆菌中表达并纯化出分子量为27kD HaPrP蛋白,Western blot证实可与PrP特异性单克隆抗体反应。将纯化蛋白在体外标记生物素(Biotin-7-NHs),与纯化的scrapie 263K毒株仓鼠感染脑组织PrP^Se蛋白共同孵育4天后,经蛋白酶K消化,Western blot检测,证明存在一条抵抗蛋白酶K消化的、被亲和素特异性识别的条带,其分子量约为20kD。这表明HaPrP^sen可以被转化为HaPrP^res。实验表明,可以利用原核系统取代真核系统表达PrP,并用生物素标记取代^35S标记纯化蛋白,建立一个更加安全方便的无细胞构象转化系统,用于朊病毒的研究。     

微管相关蛋白tau与朊蛋白的相互作用

微管相关蛋白tau参与了许多神经退行性疾病的发生, 其中包括一些人类可传播性海绵状脑病. 为了探讨tau与朊蛋白(PrP)之间可能存在的关系, 首先通过GST pull-down和免疫共沉淀等技术发现重组tau蛋白可通过微管结合区与来源于正常叙利亚仓鼠脑组织中的正常细胞膜朊蛋白(PrP^C)和羊瘙痒因子263K感染仓鼠脑组织的异常朊蛋白(PrPSc)相结合. 利用免疫共沉淀实验发现在正常和羊瘙痒因子感染的仓鼠脑组织中存在tau蛋白与PrPC和PrPSc的相互作用, 并且利用激光共聚焦方法检测到PrP和tau蛋白在CHO细胞内具有共定位的关系. 为了确定PrP与tau蛋白相互作用的部位, 构建了不同区域的PrP片段, 从而证明PrP与tau蛋白相互作用的区域位于PrP的N端序列(23~91 aa). PrP与tau蛋白分子间相互作用的直接实验证据提示tau蛋白可能参与PrP的正常生理功能以及朊病毒病的病理过程.     

感染羊瘙痒因子263K株或139A株仓鼠脑组织中tau蛋白和磷酸化tau蛋白变化的研究

为探讨朊病毒病的神经病理特征,应用Western blot方法检测了感染羊瘙痒因子263K株或139A株的仓鼠脑组织中总tau蛋白和Ser396和Ser404位点发生磷酸化tau蛋白表达水平的变化;并应用Real Time PCR方法检测了tau mRNAs转录活性的改变。结果表明总tau蛋白含量升高而Ser396和Ser404位点发生磷酸化的tau蛋白含量降低,该现象与羊瘙痒因子毒株类型和临床潜伏期无关;感染羊瘙痒因子的仓鼠脑组织中Tau2和Tau4这两个异构体的转录水平升高。这些结果表明tau蛋白在Ser396和Ser404位点的去磷酸化可能与朊病毒病发病相关。     

青藏高原藏羚羊朊蛋白基因及氨基酸序列分析

羊瘙痒病是一种自然发生的传染性海绵状脑病,它可以在绵羊和山羊羊群之中传播,其疾病的易感性、潜伏期和跨物种传播的能力主要受宿主朊蛋白基因(prion protein gene,PRNP)的影响。研究藏羚羊PRNP有助于明确藏羚羊和羊瘙痒病之间的关系,从而更好地保护藏羚羊。参照GenBank上已经发表的绵羊PRNP序列设计引物,然后对藏羚羊的PRNP序列进行扩增、测序和分析。测序结果显示藏羚羊PRNP序列由771个核苷酸构成,编码256个氨基酸。序列分析结果显示21只藏羚羊PrP(prion protein,PrP)氨基酸序列完全同源,并且与野生型绵羊PrP氨基酸序列完全一致。此外,藏羚羊PrP氨基酸序列与山羊(99.2%)、汤氏瞪羚(99.2%)、印度羚(99.2%)、驯鹿(98%)、马鹿(98%)、家牛(97.7%)和水牛(96.1%)也具有较高的同源性。在对PrP氨基酸序列136、154、171位多态性分析后发现本次收集的21只藏羚羊样本可能和野生型绵羊一样对羊瘙痒病易感。本研究为羊瘙痒病在藏羚羊羊群中可能出现的风险提供了理论依据,提示要加强藏羚羊羊群中羊瘙痒病的监测。     

疯牛病(TSE)检测的研究进展

近年来,对TSE检测的研究发展非常迅速,尽管朊病毒的分类本质尚不明确,但一些生物学特性已有研究,如传染性,抵抗力等,其检测技术包括;早期利用宿主动物牛或转基因小鼠的生物学方法;有多种免疫诊断方法可区分正常和异常的朊病毒蛋白,欧盟验证了三种免疫诊断方法,瑞士Prionics公司的免疫印迹法,爱尔兰Enfer Scientific公司的ELISA法和法国CEA和英国Bio-Rad公司开发的夹心免疫法,三种检测方法准确性为100%,因此,欧盟授权这三种检测方法用于欧洲检测疯牛病,此外,基于免疫荧光方法和毛细行之有效免疫电泳的方法也有应用;研究人员还尝试通过对TSE感染过程中出现的相关蛋白,朊病毒的形态变化的检测来诊断TSE,也有针对TSE发病过程中,异常朊病毒蛋白在脑部出现聚集,采用不同手段用以诊断;在体外模拟感染性PrP(Sc)引起正常细胞表面蛋白PrP构象改变的过程也应用于TSE的检测,对潜伏期的诊断试剂研究正在进行中,但目前并未取得满意的发出高敏感性,高特异性和使用方便的TSE诊断方法。     

朊病毒播散机制的研究进展

传染性海绵状脑病(Transmissible spongiform encephalopathies,TSE)是一类致死性中枢神经系统的慢性退化性疾病,它包括了疯牛病(Mad cowdisease,MCD)即牛海绵状脑病(Bovine spongiformencephalopathy,BSE)、羊瘙痒病(Scrapie)、克雅病(Creutzfeldt-Jakob disease,CJD)、GSS综合征     

Amphotericin B delays both scrapie agent replication and PrP-res accumulation early in infection.

Amphotericin B delays the onset of clinical symptoms in hamsters infected with scrapie agent strain 263K. Here we show that accumulation of a scrapie-specific isoform of the prion protein (PrP-res) and agent replication were delayed early in amphotericin B-treated animals. By 8 weeks postinfection, only untreated animals exhibited clinical symptoms of scrapie infection whereas PrP-res levels and titers were similar in treated and untreated animals. This suggests that although PrP-res accumulation and agent replication are linked, they are not the sole factors required for the onset of clinical disease.     

Multiple amino acid residues within the rabbit prion protein inhibit formation of its abnormal isoform

Transmissible spongiform encephalopathies (TSEs) are neurological diseases that are associated with the conversion of the normal host-encoded prion protein (PrP-sen) to an abnormal protease-resistant form, PrP-res. Transmission of the TSE agent from one species to another is usually inefficient and accompanied by a prolonged incubation time. Species barriers to infection by the TSE agent are of particular importance given the apparent transmission of bovine spongiform encephalopathy to humans. Among the few animal species that appear to be resistant to infection by the TSE agent are rabbits. They survive challenge with the human kuru and Creutzfeldt-Jakob agents as well as with scrapie agent isolated from sheep or mice. Species barriers to the TSE agent are strongly influenced by the PrP amino acid sequence of both the donor and recipient animals. Here we show that rabbit PrP-sen does not form PrP-res in murine tissue culture cells persistently infected with the mouse-adapted scrapie agent. Unlike other TSE species barriers that have been studied, critical amino acid residues that inhibit PrP-res formation are located throughout the rabbit PrP sequence. Our results suggest that the resistance of rabbits to infection by the TSE agent is due to multiple rabbit PrP-specific amino acid residues that result in a PrP structure that is unable to refold to the abnormal isoform associated with disease.     

Flexible N-terminal region of prion protein influences conformation of protease-resistant prion protein isoforms associated with cross-species scrapie infection in vivo and in vitro

Transmissible spongiform encephalopathy (TSE) diseases are characterized by the accumulation in brain of an abnormal protease-resistant form of the host-encoded prion protein (PrP), PrP-res. PrP-res conformation differs among TSE agents derived from various sources, and these conformational differences are thought to influence the biological characteristics of these agents. In this study, we introduced deletions into the flexible N-terminal region of PrP (residues 34-124) and investigated the effect of this region on the conformation of PrP-res generated in an in vitro cell-free conversion assay. PrP deleted from residues 34 to 99 generated 12-16-kDa protease-resistant bands with intact C termini but variable N termini. The variable N termini were the result of exposure of new protease cleavage sites in PrP-res between residues 130 and 157, suggesting that these new cleavage sites were caused by alterations in the conformation of the PrP-res generated. Similarly truncated 12-16-kDa PrP bands were also identified in brain homogenates from mice infected with mouse-passaged hamster scrapie as well as in the cell-free conversion assay using conditions that mimicked the hamster/mouse species barrier to infection. Thus, by its effects on PrP-res conformation, the flexible N-terminal region of PrP seemed to influence TSE pathogenesis and cross-species TSE transmission.     

Immunological comparison of scrapie-associated fibrils isolated from animals infected with four different scrapie strains.

Scrapie-associated fibrils (SAFs) are abnormal filamentous structures that are uniquely associated with unconventional slow virus diseases. The antigenic relationships of SAFs from animals infected with four biologically distinct scrapie strains were investigated by using antisera raised to purified SAF proteins. Rabbit antisera were raised to SAFs isolated from mice infected with the ME7 scrapie strain and to SAFs isolated from hamsters infected with the 263K scrapie strain. A strong antigenic relationship was shown among SAF proteins (PrPs) isolated from all scrapie-infected animals (ME7, 139A, and 87V in mice and 263K in hamsters), and this relationship was demonstrable regardless of which antiserum was used. SAF proteins were antigenically distinct from those of paired helical filaments or amyloid isolated from patients with Alzheimer disease. Distinct Western blot profiles were demonstrated for SAFs isolated from animals infected with each scrapie strain. Differences seen among SAFs were independent, at least in part, of host species or genotype, implying that certain specific structural and molecular properties of SAFs are mediated by the strain of scrapie agent.     

Molecular basis of scrapie strain glycoform variation

Transmissible spongiform encephalopathies (TSE) are characterized by the conversion of a protease-sensitive host glycoprotein, prion protein or PrP-sen, to a protease-resistant form (PrP-res). PrP-res molecules that accumulate in the brain and lymphoreticular system of the host consist of three differentially glycosylated forms. Analysis of the relative amounts of the PrP-res glycoforms has been used to discriminate TSE strains and has become increasingly important in the differential diagnosis of human TSEs. However, the molecular basis of PrP-res glycoform variation between different TSE agents is unknown. Here we report that PrP-res itself can dictate strain-specific PrP-res glycoforms. The final PrP-res glycoform pattern, however, can be influenced by the cell and significantly altered by subtle changes in the glycosylation state of PrP-sen. Thus, strain-specific PrP-res glycosylation profiles are likely the consequence of a complex interaction between PrP-res, PrP-sen, and the cell and may indicate the cellular compartment in which the strain-specific formation of PrP-res occurs.     

Remarkable reduction of MAP2 in the brains of scrapie-infected rodents and human prion disease possibly correlated with the increase of calpain

Microtubule-associated protein 2 (MAP2) belongs to the family of heat stable MAPs, which takes part in neuronal morphogenesis, maintenance of cellular architecture and internal organization, cell division and cellular processes. To obtain insight into the possible alteration and the role of MAP2 in transmissible spongiform encephalopathies (TSEs), the MAP2 levels in the brain tissues of agent 263K-infected hamsters and human prion diseases were evaluated. Western blots and IHC revealed that at the terminal stages of the diseases, MAP2 levels in the brain tissues of scrapie infected hamsters, a patient with genetic Creutzfeldt-Jakob disease (G114V gCJD) and a patient with fatal familial insomnia (FFI) were almost undetectable. The decline of MAP2 was closely related with prolonged incubation time. Exposure of SK-N-SH neuroblastoma cell line to cytotoxic PrP106-126 peptide significantly down-regulated the cellular MAP2 level and remarkably disrupted the microtubule structure, but did not alter the level of tubulin. Moreover, the levels of calpain, which mediated the degradation of a broad of cytoskeletal proteins, were significantly increased in both PrP106-126 treated SK-N-SH cells and brain tissues of 263K prion-infected hamsters. Our data indicate that the decline of MAP2 is a common phenomenon in TSEs, which seems to occur at an early stage of incubation period. Markedly increased calpain level might contribute to the reduction of MAP2.     

Inhibition of protease-resistant prion protein accumulation in vitro by curcumin

Inhibition of the accumulation of protease-resistant prion protein (PrP-res) is a prime strategy in the development of potential transmissible spongiform encephalopathy (TSE) therapeutics. Here we show that curcumin (diferoylmethane), a major component of the spice turmeric, potently inhibits PrP-res accumulation in scrapie agent-infected neuroblastoma cells (50% inhibitory concentration, approximately 10 nM) and partially inhibits the cell-free conversion of PrP to PrP-res. In vivo studies showed that dietary administration of curcumin had no significant effect on the onset of scrapie in hamsters. Nonetheless, other studies have shown that curcumin is nontoxic and can penetrate the brain, properties that give curcumin advantages over inhibitors previously identified as potential prophylactic and/or therapeutic anti-TSE compounds.     

Conversion of raft associated prion protein to the protease-resistant state requires insertion of PrP-res (PrP(Sc)) into contiguous membranes

Prion protein (PrP) is usually attached to membranes by a glycosylphosphatidylinositol-anchor that associates with detergent-resistant membranes (DRMs), or rafts. To model the molecular processes that might occur during the initial infection of cells with exogenous transmissible spongiform encephalopathy (TSE) agents, we examined the effect of membrane association on the conversion of the normal protease-sensitive PrP isoform (PrP-sen) to the protease-resistant isoform (PrP-res). A cell-free conversion reaction approximating physiological conditions was used, which contained purified DRMs as a source of PrP-sen and brain microsomes from scrapie-infected mice as a source of PrP-res. Interestingly, DRM-associated PrP-sen was not converted to PrP-res until the PrP-sen was either released from DRMs by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). PEG-assisted conversion was optimal at pH 6--7, and acid pre-treating the DRMs was not sufficient to permit conversion without PI-PLC or PEG, arguing against late endosomes/lysosomes as primary compartments for PrP conversion. These observations raise the possibility that generation of new PrP-res during TSE infection requires (i) removal of PrP-sen from target cells; (ii) an exchange of membranes between cells; or (iii) insertion of incoming PrP-res into the raft domains of recipient cells.     

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.