Clinicopathologic characteristics of sporadic Japanese Creutzfeldt–Jakob disease classified according to prion protein gene polymorphism and prion protein type

We analyzed neuropathologic features of 23 Japanese patients with sporadic Creutzfeldt–Jakob disease (sCJD) by means of prion protein (PrP) immunolabeling associated with codon 129 polymorphism of the PrP gene and western blot analysis of protease-resistant PrP (PrP type). Clinical features, particularly age at onset, disease duration, periodic synchronous discharge and presence of myoclonus, were also analyzed. This study included 11 cases of subacute spongiform encephalopathy (SSE), 10 cases of panencephalopathic (PE)-type sCJD and two cases of thalamic-type sCJD, classified according to cerebral pathology findings. According to PrP gene polymorphism and PrP type, 18 cases were classified as MM1-type, two as MV1-type, two as MM2-type and one as MM1 + 2-type sCJD. SSE and PE-type sCJD showed similar clinical features, with the exception of disease duration, codon 129 polymorphism and PrP type. Thalamic-type sCJD showed different clinical features and PrP type. We suggest that SSE and PE-type sCJD comprise the sCJD subtype and that PE-type sCJD is a prolonged pathologic phenotype of SSE. When we compare our results with those from a series of Caucasian sCJD patients, the percentages of codon 129 polymorphisms differed, as did classification based on PrP gene polymorphism and PrP type; our series included many PE-type sCJD cases and disease duration was relatively long and MM2-type cases showed clinicopathologic variability.     

Dynamic Analyses of PrP and PrPsc in Brain Tissues of Golden Hamsters Infected With Scraple Strain 263K Revealed Various PrP Forms

Objective To expatiate dynamic changes in hamsters infected with scrapie strain 263K, to observe the presence and aggravation of various forms of PrP and PrPSc during incubation period, and to probe primarily the relationship between the onset of clinic manifestations and the presence of different PrPSc forms. Methods Hamster-adapted scrapie strain 263K was intracerebrally inoculated into hamsters. Different forms of PrP and PrPSc were monitored dynamically by Western blot and immuno-histochemical assays. The presence of scrapie-associated fibril (SAF) was assayedby electron microscopy analysis (EM) and immuno-golden EM. Results PrPSc was initiallydetected in the brain tissues of the animals in 20 days post-inoculation by immunohistochemistry and 40 days with Western blot. Quantitative evaluations revealed that the amounts of PrP and PrPSc inbrain tissues increased along with the incubation. Several high and low molecular masses of PrP wereseen in the brains of the long-life span infected animals. Deglycosylation assays identified that the truncated PrP in the infected brains showed similar glycosylation patterns as the full-length PrP. The presence of short fragments was seemed to relate with the onset of clinical conditions. Conclusion These results indicate that infectious agents exist and accumulate in central nerve system prior to the onset of the illness. Various molecular patterns of PrPSc may indwell in brain tissues during the infection.     

Expression profiles of prion and doppel proteins and of their receptors in mouse splenocytes

Doppel (Dpl) shares common structural features with the prion protein (PrP) whose pathologic isoform is considered as the causative agent of prion diseases. Although their physiological functions in the immune system remain largely unknown, we demonstrated that substantial amounts of PrP and Dpl are expressed by spleen cells notably B lymphocytes, granulocytes and DC, but not T lymphocytes and NK. To characterize trans-interacting partners of PrP and Dpl on mouse splenocytes, fluorescent PrP and Dpl tetramers were produced and used as tracers. Both tetramers specifically bind to B lymphocytes, dendritic cells, macrophages and granulocytes and in a lesser extend to T lymphocytes. No binding was observed on NK, follicular dendritic cells and mesenchymal spleen cells. The activation of intracellular transduction signals (i.e. intracellular calcium concentration and activation of the MAP kinase pathway) suggested that PrP and Dpl tetramers bind to functional receptors on B cells. None of the previously described PrP partners account for the binding sites characterized here. Our study suggests a possible role for PrP and Dpl in the cell-cell interactions in the immune system.     

In vivo prion protein intestinal uptake in fish

Intestinal uptake of abnormal prion protein (PrPSc), the pathological agent involved in transmissible spongiform encephalopathies (TSEs), has been investigated in rainbow trout (Oncorhynchus mykiss). Experimental procedures were conducted in vivo by immunohistological PrPSc localization in intestine and pyloric caeca after forced feeding of infected material. Results indicate that PrPSc was absorbed by the intestinal mucosa and that it persisted in the fish gastrointestinal tract for up to 3 days in pyloric caeca and for up to 7 days in the distal intestine. It did not remain longer than 15 days in the fish intestine; furthermore, it did not cross the intestinal barrier.     

Polymorphisms of the prion protein gene in sheep of Inner Mongolia,China

Polymorphisms of the prion protein gene (Prnp), especially the amino acid residue alterations at codons 136, 154, and 174, in sheep have been found to be associated with susceptibility to scrapie disease. We investigated Prnp polymorphisms in three local sheep breeds in Inner Mongolia, China. Blood samples were collected from 46 Ujumqin, 34 Sunite, and 22 Mongolian sheep. The genetic DNA of blood samples was extracted, amplified and sequenced, and amino acid alignment was determined. Polymorphisms were detected at 8 codons, among which M157I, Q220H, and R223K have not been previously reported. The frequency of the amino acid residues ARQ/ARQ at codons 136, 154, and 171, respectively, which is associated with medium-high susceptibility to scrapie, was 74.5%, and the frequency of scrapie-resistant genotype ARR/ARR was 7.9%. The highly susceptible genotype VRQ/VRQ at these codons as not detected from the tested sheep. Of the three sheep breeds, Ujumqin sheep had the highest frequency (15.2%) of scrapie-resistant amino acid sequence, ARR/ARR at codons 136, 154, and 171, respectively, accounting for 87.5% sheep that carry these polymorphisms. Our findings are of special importance for both live sheep export and sheep breeding.     

Characterisation of new monoclonal antibodies reacting with prions from both human and animal brain tissues

Post-mortem diagnosis of transmissible spongiform encephalopathies (prion diseases) is primarily based on the detection of a protease resistant, misfolded disease associated isoform (PrP(Sc)) of the prion protein (PrP(C)) on neuronal cells. These methods depend on antibodies directed against PrP(C) and capable of reacting with PrP(Sc)in situ (immunohistochemistry on nervous tissue sections) or with the unfolded form of the protein (western and paraffin embedded tissue (PET) blotting). Here, high-affinity monoclonal antibodies (mAbs 1.5D7, 1.6F4) were produced against synthetic PrP peptides in wild-type mice and used for western blotting and immunohistochemistry to detect several types of human prion-disease associated PrP(Sc), including sporadic Creutzfeldt-Jakob Disease (CJD) (subtypes MM1 and VV2), familial CJD and Gerstmann-Str?ussler-Scheinker (GSS) disease PrP(Sc) as well as PrP(Sc) of bovine spongiform encephalopathy (bovine brain), scrapie (ovine brain) and experimental scrapie in hamster and in mice. The antibodies were also used for PET-blotting in which PrP(Sc) blotted from brain tissue sections onto a nitrocellulose membrane is visualized with antibodies after protease and denaturant treatment allowing the detection of protease resistant PrP forms (PrP(RES)) in situ. Monoclonal antibodies 1.5D7 and 1.6F4 were raised against the reported epitope (PrP153-165) of the commercial antibody 6H4. While 1.5D7 and 1.6F4 were completely inhibitable by PrP153-165, 6H4 was not, indicating that the specificity of 6H4 is not defined completely by PrP153-165. The two antibodies performed similarly to 6H4 in western blotting with human samples, but showed less reactivity and enhanced background staining with animal samples in this method. In immunohistochemistry 1.5D7 and 1.6F4 performed better than 6H4 suggesting that the binding affinity of 1.5D7 and 1.6F4 with native (aggregated) PrP(Sc)in situ was higher than that of 6H4. On the other hand in PET-blotting, 6H4 reached the same level of reactivity as 1.5D7 and 1.6F4. This shows that 6H4 needs denatured PrP(RES) to reach maximal reactivity, confirming earlier results. As an exception, human PrP(RES) still reacted relatively poorly with 6H4 in PET-blotting, while 1.5D7 and 1.6F4 reacted well with PrP(RES) from most human CJD types. Taken together this implies that the binding epitope of 1.5D7 and 1.6F4 is accessible in the aggregates of undenatured PrP(Sc) (IHC) while the binding site of 6H4 is at least partly inaccessible. In techniques incorporating a denaturing and/or disaggregating step 6H4 showed good binding indicating increased accessibility of the binding site. An exception to this is human samples in PET-blotting suggesting that huPrP(RES) might not be as easily unfolded by denaturation as BSE and scrapie PrP(RES). Also of interest was the ability of 1.5D7 and 1.6F4 to discriminate between two allelic variants of PrP CJD(Sc) (VV vs. MM) in immunohistochemistry as opposed to the normally used antibody 3F4.     

SIRT1, a histone deacetylase, regulates prion protein-induced neuronal cell death

Prion diseases associated with the conversion of the cellular prion protein (PrP(C)) to the misfolded isoform (PrP(Sc)), affect the central nervous system (CNS) of humans and animals. Resveratrol, an activator of class III histone deacetylase SIRT1, is important in attenuating cellular injury and oxidative stress. The present study investigated the effects of SIRT1 activation on prion protein-mediated neuronal cell death and examined its possible signals in intracellular apoptotic pathways. Resveratrol treatment significantly increased both SIRT1 protein expression and SIRT1 activity and protected neuronal cells against PrP (106-126)-induced cell death. Resveratrol-mediated SIRT1 activation decreased the acetylation of p53 and p65 induced by prion protein and SIRT1 inhibitor. SIRT1 activation also inhibited PrP (106-126)-mediated p38 mitogen-activating protein kinase (MAPK) activation and caspase-3 cleavage, and increased the expression of anti-apoptotic Bcl-xL protein. Furthermore, SIRT1 overexpression by using adenoviral vector protected neuronal cells against PrP (106-126). These results indicate that resveratrol inhibits PrP (106-126)-induced neuronal cell death by regulating SIRT1 activity and SIRT-related signaling, and suggest that prion-related disease may be attenuated by SIRT1 activation or by intake of SIRT1-activating molecules.     

APP695K595N/M596L突变转基因小鼠的建立和病理表型动态分析

目的建立APP695K595N/M596L(Swedish突变)转基因小鼠和评价痴呆表型的发生和发展过程。方法将APP695K595N/M596L突变基因插入到小鼠朊蛋白(mouse prion protein)启动子下游,构建转基因表达载体,通过显微注射法建立APP695K595N/M596L突变转基因C57BL/6J小鼠。PCR鉴定转基因小鼠的基因表型,Western blotting检测APP突变基因表达。Thioflavin-S染色检测不同年龄转基因小鼠大脑病理改变。Morris水迷宫动态观察小鼠行为学改变。结果建立了人APP695K595N/M596L转基因小鼠,Thioflavin-S染色显示转基因小鼠9月龄时在脑海马区可检测到老年斑形成,并且在11、12月龄时明显增多。Morris水迷宫结果发现与同月龄野生型小鼠相比,该转基因小鼠5月龄开始出现学习记忆能力缺陷,7、9、11月行为学结果证实转基因小鼠的学习记忆能力缺陷随年龄增加而日趋严重(P<0.05)。结论建立了人APP695K595N/M596L转基因小鼠,并能再现人类阿尔茨海默症的行为学及神经病理学特征,为阿尔茨海默病发病机制研究和药物研发提供了有价值的动物模型。     

Synthesis of 9-substituted 2,3,4,9-tetrahydro-1H-carbazole derivatives and evaluation of their anti-prion activity in TSE-infected cells

2,3,4,9-Tetrahydro-9-[2-hydroxy-3-(1-piperidinyl)propyl]-6-methyl-1H-carbazol-1-one (GJP14) is a novel anti-prion compound that we previously discovered by in silico screening and cellular assay. In this study, a variety of GJP14 derivatives were prepared using pyrrole derivatives, (haloalkyl)oxiranes, and amines, and their anti-prion activity was evaluated in TSE-infected cells. It was found that the tricyclic aromatic ring, a hydroxy group at the 2-position and an amino group at the 3-position of the N-propyl group were the basic requirements for anti-prion activity. The derivatives bearing an N-ortho-halobenzyl group exhibited an improved activity, and the most potent derivative was 8 times as effective as the original lead compound, GJP14.