Evaluation of the effect of various main elements on the PrPSc detection by real-time quaking-induced conversion assay.

In order to definitively diagnosis sporadic Creutzfeldt­Jakob disease (sCJD), brain tissue is currently required. Therefore, there is a great need for tests that can detect sCJD in body fluids or other types of tissues. Different variables, including the amount of recombinant celluar prion protein (rPrPC), salt, cleaning surfactants and thioflavin T (ThT), in human cerebrospinal fluid (CSF) were evaluated. The reagent concentrations of 1X PBS, 170 mM NaCl, 1 mM EDTA, 0.01 mM ThT and 0.001% SDS, and the amounts of 10 µg rPrPC and 10 µl CSF were considered to be optimal for the real­time quaking­induced conversion (RT­QuIC) assay. Using these conditions, the RT­QuIC assay for prion protein (PrPSc) detection was observed to be sensitive to 10­8 diluted brain homogenates of hamsters infected with the 263K scrapie strain. Furthermore, CSF samples from 70 probable sCJD cases and 48 non­CJD cases were preliminarily screened. A substantial proportion of sCJD samples (57.14%) tested positive by RT­QuIC, with a short lag phase (<50 h post­reaction) and high peak ThT values (>25,000 relative fluorescence units). By contrast, only a small number of non­CJD samples displayed weakly positive results, and these were detected at a later stage (>50 h post­reaction) and had much lower ThT values. In conclusion, the RT­QuIC assay in CSF samples reported in the present study may provide a useful pre­mortem tool for the diagnosis of sCJD, particularly in China where postmortem examination is rarely conducted.

Redox induces diverse effects on recombinant human wild-type PrP and mutated PrP with inserted or deleted octarepeats.

Normal prion protein (PrP) contains two cysteines at amino acids 179 and 214, which may form intra­ and interpeptide disulfide bonds. To determine the possible effects of this disulfide bridge on the biochemical features of PrP, prokaryotic recombinant human wild­type PrP (PG5), and mutated PrPs with seven extra octarepeats (PG12) or with all five octarepeats removed (PG0), were subjected to redox in vitro. Sedimentation assays revealed a large portion of aggregation in redox­treated PG5, but not in PG0 and PG12. Circular dichroism analysis detected increased ß­sheet and decreased α­helix in PG5 subjected to redox, increased random­coil and decreased ß­sheet in PG0, and increased random­coil, but limited changes to ß­sheet content, in PG12. Thioflavin T fluorescence tests indicated that fluorescent value was increased in PG5 subjected to redox. In addition, proteinase K (PK) digestions indicated that PK resistance was stronger in PG12 and PG0 compared with in PG5; redox enhanced the PK resistance of all three PrP constructs, particularly PG0 and PG12. These data indicated that formation of a disulfide bond induces marked alterations in the secondary structure and biochemical characteristics of PrP. In addition, the octarepeat region within the PrP peptide markedly influences the effects of redox on the biochemical phenotypes of PrP, thus highlighting the importance of the number of octarepeats in the biological functions of PrP.

Reduction of NF-κB (p65) in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents.

Transcription factor NF-κB functions as a pleiotropic regulator of target genes controlling physiological function as well as pathological processes of many different diseases, including some neurodegenerative diseases. However, the role of NF-κB in the pathogenesis of prion disease remains ambiguous. In this study, the status of NF-κB (p65) in a prion-infected cell line SMB-S15 was first evaluated. Significantly lower levels of p65 and the phosphorylated form of p65 (p-p65) were detected in SMB-S15 cells, compared with its normal partner cell line SMB-PS. Markedly slower responses of the NF-κB system to the stimulation of TNF-α were observed in SMB-S15 cells. Removal of PrPSc replication in SMB-S15 cells rescued the expression and activity of NF-κB. However, overexpression of p65 in SMB-S15 cells did not influence the propagation of PrPSc. Moreover, significant decline of p65 level was also observed in the brain tissues of mice infected with the lysates of SMB-S15 cells and hamsters infected with scrapie agent 263K at terminal stage. Immunofluorescence assays (IFAs) on brain sections from either normal or scrapie-infected rodents revealed colocalization of p65 with neuronal nuclear (NeuN) protein positive cells but not with glial fibrillary acidic protein (GFAP) positive cells. Assays of the agents involving in the regulation of NF-κB showed down-regulated phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB/Akt) both in SMB-S15 cells and in the brains of scrapie-infected rodents. Those data indicate a remarkable repression of the classical NF-κB pathway during prion infection both in vitro and in vivo. The alteration of NF-κB (p65) shows close association with the replication and accumulation of PrPSc in the cells.

Clinical and laboratory features of 14 young Chinese probable sCJD patients.

Sporadic Creutzfeldt-Jakob disease (sCJD) occurs frequently in the relatively older population, mainly in the groups of 60-69 and 70-79 year-old. Since 2006 when China performed national CJD surveillance, 14 young probable sCJD patients below 40 year-old were identified, counting for 1.93% of all probable sCJD cases. The clinical features of young probable sCJD cases, including the onset feature, the presence of sCJD-associated signs and the clinical duration, are indistinguishable from those of older patients. Special sCJD-associated abnormalities on EEG and MRI were noticed in 7 and 10 cases. CSF 14-3-3 was positive in 7 cases. CSF RT-QuIC showed positive reactive curves in 9 cases, with short lag phases. PRNP sequencing did not find any mutation. Due to low rate of brain autopsy in China, performances of other CJD-associated examinations as much as possible are extremely important for the distinguish diagnosis of young probable sCJD patients.

The Brain NO Levels and NOS Activities Ascended in the Early and Middle Stages and Descended in the Terminal Stage in Scrapie-Infected Animal Models.

The infections of prion agents may cause progressive and fatal neurodegenerative diseases in humans and a serial of animal species. Previous studies have proposed that the levels of nitric oxide (NO) and nitric oxide synthase (NOS) in the brains of some neurodegeneration diseases changed, while S-nitrosylation (SNO) of many brain proteins altered in prion diseases. To elucidate the potential changes of brain NO levels during prion infection, the NO levels and NOS activities in the brain tissues of three scrapie experimental rodents were measured, including scrapie agent 263 K-infected hamsters and 139A- and ME7-infected mice. Both NO levels and NOS activities, including total NOS (TNOS) and inducible NOS (iNOS), were increased at the terminal stages of scrapie-infected animals. Assays of the brain samples collected at different time points during scrapie infection showed that the NO levels and NOS activities started to increase at early stage, reached to the peak in the middle stage, and dropped down at late stage. Western blots for brain iNOS revealed increased firstly and decreased late, especially in the brains of 139A- and ME7-infected mice. In line with those alterations, the levels of the SNO forms of several selected brain proteins such as aquaporin-1 (AQP1), calcium/calmodulin-dependent protein kinase II (CaMKII), neurogranin, and opalin, underwent similar changing trends, while their total protein levels did not change obviously during scrapie infection. Our data here for the first time illustrate the changing profile of brain NO and NOS during prion infection. Time-dependent alterations of brain NO level and the associated protein S-nitrosylation process may contribute greatly to the neuropathological damage in prion diseases.

Comparative proteomics analyses for 139A and ME7 scrapie infected mice brains in the middle and terminal stages.

PURPOSE: To analyze the proteomics patterns in the cortex regions of scrapie strains 139A- and ME7-infected mice collected in the middle and terminal stages. EXPERIMENTAL DESIGN: Western Blot and immunohistochemistry methods are used to analyze the pathological changes in mice collected in the middle and terminal stages. The technique of iTRAQ and multidimensional LC and MS are used to analyze the proteomics patterns of mice in different stages. RESULTS: In total, 2891 with 95% confidence interval are identified. The study here also demonstrates a similar protein expressions in the CNS tissues of two scrapie strains infected mice at the terminal stages, but markedly different one between the middle and terminal samples, not only in the numbers of differentially expressed proteins and involved gene ontologies and pathways but also in the relevant functional constitutions. CONCLUSIONS: It may provide useful clue in exploring the abnormalities of biological functions at different time points of prion infections and in searching for potential therapeutic and diagnostic biomarkers for prion diseases.

Protein Misfolding Cyclic Amplification Cross-Species Products of Mouse-Adapted Scrapie Strain 139A and Hamster-Adapted Scrapie Strain 263K with Brain and Muscle Tissues of Opposite Animals Generate Infectious Prions.

Transmission of prions between mammalian species is limited by a "species barrier," a biological effect involving an increase in incubation period to decrease the percentage of animals succumbing to disease. In this study, we used protein misfolding cyclic amplification (PMCA) technique, which accelerates the conversion of prion proteins in vitro. Direct interspecies PMCA involving 144 cycles confirmed that both mouse-adapted scrapie strain 139A and hamster-adapted 263K could use brain homogenates of opposite species to form proteinase K (PK)-resistant PrP proteins (PrPres). Newly formed interspecies prions could stably propagate themselves in subsequent serial PMCA passages. The two types of PMCA-generated cross-species PrPres changed their glycosylation profiles, which was similar to that observed during interspecies infection by the mouse agent 139A in vivo. These profiles were distinct from individual seeded PrPSc and possessed properties of new hosts. Comparative analysis with respect to PK resistance showed no significant diversity between PMCA-PrPres and native PrPSc or between brain and muscle PrPres. However, PrPres from the relatively early cycles of serial PMCA showed lower PK resistance than those from later cycles. Inoculation of these PMCA products amplified with homogeneous or heterogeneous brain tissues (cross-species products) induced experimental transmissible spongiform encephalopathies. These results suggested that PMCA can help prion strains to overcome species barrier and to propagate efficiently both in vitro and in vivo.

MiRNA expression profiles in the brains of mice infected with scrapie agents 139A, ME7 and S15.

MicroRNA (miRNA) is a class of non-coding endogenous small-molecule single-stranded RNA that regulates complementary mRNA through degradation or translation of the mRNA targets. Usually, miRNAs show remarkable cell and tissues specificity. Recently, alterations in a set of miRNAs in the brains of patients with certain neurodegenerative diseases, including prion diseases, have been reported. In this study, using deep sequencing technology, miRNA expression profiles in the brains of mice infected with scrapie agents 139A, ME7 and S15 at a terminal stage were comparatively analysed. In total, 57, 94 and 135 differentially expressed miRNAs were identified in the pooled brain samples of 139A-, ME7- and S15-infected mice, respectively, compared with the brains of age-matched normal controls. Among them, 22 were commonly increased and 14 were commonly decreased in the brains of all three infected models. In addition, a reduction in the expression of two novel miRNAs was also commonly observed. Quantitative PCR with reverse transcription analysis of six randomly selected commonly increased and decreased miRNAs in the brains of the three infected mouse models, as well as the two novel miRNAs, verified that the expression patterns were comparable to the deep sequencing data. KEGG analysis of the differentially expressed miRNAs revealed the involvement of similar pathways in all three types of infected animals. Comprehensive analysis of these miRNA profiles not only provides useful clues for understanding prion biology but also is beneficial in the search for possible diagnostic marker(s) for prion diseases.

Quality evaluation for the surveillance system of human prion diseases in China based on the data from 2010 to 2016.

The surveillance of CJD or human prion diseases (PrDs) has been conducted for 10 y in China. To evaluate the quality of China CJD surveillance system, the collections of the clinical and epidemiological information, the sampling, the clinical examinations and laboratory tests and follow-up survey were separately analyzed based on the data from 2010 to 2015. The obtaining rates of clinical-information table, epidemiological-information table, sample inspection sheet and medical record of the referring patients from reporting units to the center of CJD surveillance maintained or reached at very high levels, being close to 100% in the past 3 y. 93.82%, 85.23%, 96.21% and 94.70% of the reported cases had the data of MRI, EEG, CSF 14-3-3 and PRNP sequencing, respectively. Follow-up surveys were conducted in about 50% cases in 2010 and 2011, 93.39% cases in 2012 and 100% cases in the last 3 y. High obtaining rates of the clinical and epidemiological data, high conducting rates of the relevant clinical examinations and laboratory tests, high performing rates of follow-up survey for every referring case reflect a good implemental capacity in China CJD surveillance system, which supplies solid basis for recognition and diagnosis of human prion diseases and guarantees good quality of China CJD surveillance system.

Application of epigenetic markers in molecular breeding of the swine.

Livestock phenotypes are determined by the interaction of a variety of factors, including the genome, the epigenome and the environment. Epigenetics refers to gene expression changes without DNA sequence alterations. Epigenetic markers mainly include DNA methylation, histone modifications, non-coding RNAs, and imprinting genes. More and more researches show that epigenetic markers play an important role in the traits of pigs by modulating phenotype changes via gene expression. However, the role of epigenetic markers has caught little attention in swine breeding. The mechanism that influences important traits of swine has not been analyzed in detail, and it still lacks adequate scientific basis for practical applications. From the aspects of nutrition, diseases, important economic traits and trans-generational inheritance, we summarize the research, application prospects and challenges in the field of utilizing epigenetic markers in molecular breeding of pigs, thus providing a more comprehensive theoretical basis to promote more rapid research development in this field.

Down-regulation of brain-derived neurotrophic factor and its signaling components in the brain tissues of scrapie experimental animals.

Prion is a unique nucleic acid-free pathogen that causes human and animal fatal neurodegenerative diseases. Brain-derived neurotrophic factor (BDNF) is a prototypic neurotrophin that helps to support the survival of existing neurons, and encourage the growth and differentiation of new neurons and synapses through axonal and dendritic sprouting. There are two distinct classes of glycosylated receptors, neurotrophin receptor p75 (p75NTR) and tropomyosin-related kinase (Trk), that can bind to BDNF. To obtain insights into the possible alterations of brain BDNF and its signaling pathway in prion disease, the levels of BDNF and several molecules in the BDNF pathway in the brain tissues of scrapie agents 263K-infected hamsters were separately evaluated. Western blots and/or immunohistochemical (IHC) assays revealed that BDNF, TrkB, GRB2 and p75NTR, were significantly downregulated in the brain tissues of scrapie-infected rodents at terminal stage. Double-stained immunofluorescent assay (IFA) demonstrated that BDNF and phospho-TrkB predominately expressed in neurons. Dynamic analyses of the brain samples collected at the different time-points during the incubation period illustrated continuous decreases of BDNF, TrkB, phospho-TrkB, GRB2 and p75NTR, which correlated well with neuron loss. However, these proteins remained almost unchanged in the prion infected cell line SMB-S15 compared with those of its normal cell line SMB-PS. These data suggest that the BDNF signaling pathway is severely hindered in the brains of prion disease, which may contribute, at least partially, to the neuron death.

Rare E196A mutation in PRNP gene of 3 Chinese patients with Creutzfeldt-Jacob disease.

Inherited prion diseases are characterized by mutations in the PRNP gene, which account for 5-15% of human prion diseases. Here we reported 3 Chinese genetic Creutzfeldt-Jacob disease cases (gCJD) with a rare mutation in PRNP leading to an exchange of amino acid from glutamic acid (E) to alanine (A) at codon 196 (E196A). All three patients were Han Chinese without any sibship among them. They showed various unspecific symptoms at onset and displayed typical clinical manifestations of sporadic CJD with progress of disease. The same time, 2 cases showed psychotic symptoms during the clinical courses. 14-3-3 proteins were positive in cerebrospinal fluid (CSF) and special abnormality were detected in MRI of all the cases. The polymorphism of codon 129 was methionin homozygote and that of codon 219 was glutamate homozygote in all 3 patients. The disease durations of the 3 cases varied from 10 to 22 months and no disease associated family history was figured out in all the cases.

[A mechanistic review of how hypoxic mircroenvironment regulates mammalian ovulation].

Mammalian ovulation is a complicated process that includes development of follicles, ovulation, formation of corpus luteum and luteolysis. The three different stages of the ovulation activity are affected by hypoxic microenvironment and hypoxia-induced factors (HIF), which play a crucial role in physiologyical processes, such as angiogenesis and inflammation. Although the process of ovulation has been well elucidated, the molecular mechanism regulated by hypoxia needs an in depth study. In this review, we summarize how hypoxic and HIF regulate gene expression during mammalian ovulation in order to provide a better understanding of ovulation mechanism, which may lay a theoretical basis for prevention and therapy of various ovarian diseases.

Re-infection of the prion from the scrapie­infected cell line SMB-S15 in three strains of mice, CD1, C57BL/6 and Balb/c.

It is well known that the SMB-S15 cell line was originally established by cultures from the brains of mice affected by the Chandler scrapie strain, and this cell line may express PrPSc permanently. However, the infectivity of the S15-derived prions on experimental animals has not yet been well documented. In the present study, the cell lysates of SMB-S15 were intracerebrally inoculated into three different strains of mice, namely C57BL/6, Balb/c and CD1. Prion protein (PRNP) gene sequencing revealed the same encoded PrP proteins in the sequences of amino acids in the three strains of mice, in addition to a synonymous single nucleotide polymorphism (SNP) in CD1 mice. All infected mice developed typical experimental transmissible spongiform encephalopathies (TSEs) approximately six months post-infection. The clinical features of three infected mice were comparable. The pathogenic characteristics, such as the electrophoretic and glycosylation profiles and proteinase K (PK) resistance of PrPSc molecules, as well as the neuropathological characteristics, such as spongiform vacuolation, PrPSc deposits in cortex regions, astrogliosis and activated microglia, were also similar in all three strains of infected mice. However, PrPSc deposits in the cerebellums of CD1 mice were significantly fewer, which was linked with the observation that lower numbers of CD1 mice presented cerebellum-associated symptoms. Successive inoculation of the individual strains of mice with brain homogenates from the infected mice also induced typical experimental scrapie. The data in the present study thus confirm that the prion agent in SMB-S15 cells causes stable infectivity in different types of mice with distinct phenotypes after long-term propagation in vitro. The present study also provides further scrapie rodent models, which may be used in further studies.

[The mechanism of miRNA-mediated PGR signaling pathway in regulating female reproduction].

MicroRNAs (miRNAs) are involved in several physiological processes as important post-transcriptional regulators. Progesterone (P4), an important steroid hormone, produces physiological effect through binding specific receptor progesterone receptors (PGR) which regulates functions of both reproductive and non-reproductive tissues as a member of the nuclear receptor superfamily. P4/PGR and miRNAs could regulate female reproduction independently, however, it is still unclear how miRNAs and P4/PGR interaction regulates female reproductive activities such as ovulation in female reproduction. In this review, we summarize the possible ways in which miRNAs regulate P4 production and PGR gene expression as well as P4/PGR regulate miRNAs expression, which provide a theoretical basis for further studying the role of miRNAs and P4/PGR in female reproduction.

Treatment of SMB-S15 Cells with Resveratrol Efficiently Removes the PrP(Sc) Accumulation In Vitro and Prion Infectivity In Vivo.

Prion diseases are transmissible and invariably fatal neurodegenerative disorders, which still lack of efficacious prophylactic and therapeutic tools. Our previous study has proposed that the natural phytoalexin, resveratrol, can reduce the amounts of PrP(Sc) in a scrapie-infected cell line SMB-S15. To address its anti-prion efficacy, the inhibitive activity of resveratrol on prion accumulation in vitro and prion infectivity in vivo was analyzed in the present study. Exposure of SMB-S15 cells to various concentrations of resveratrol (0.25 to 200 µM) reduced and even removed cellular PrP(Sc) in a dose-dependent manner, with EC50 0.61 µM. Meanwhile, PrP(Sc) signals in SMB-S15 cells treated with 5 and 10 µM resveratrol maintained undetectable after drug withdrawal, indicating that the removal of PrP(Sc) in SMB-S15 cells by resveratrol is irreversible. Furthermore, the lysates of SMB-S15 cells exposed to 10 µM resveratrol for 2 and 7 days were intracerebrally inoculated into CD1 mice. All mice (n = 9) infected with SMB-S15 cells without treatment of resveratrol appeared typical experimental scrapie symptoms from 155 to 228 day post inoculation (dpi), while all mice (n = 9) inoculated with SMB-S15 cells treated with resveratrol for 7 days maintained healthy by the end of observations (284 dpi). PrP-specific Western blots and neuropathological tests did not identify PrP(Sc) or prion disease-associated pathological abnormality in the brains of mice inoculated with 7-day resveratrol-treated SMB-S15 cells. It indicates that the prion infectivity of SMB-S15 onto CD1 mice is eradicated by 1-week resveratrol treatment. Sensitivity of PrP(Sc) to resveratrol highlights its potential role in prion therapeutics.

Proteomic Analyses for the Global S-Nitrosylated Proteins in the Brain Tissues of Different Human Prion Diseases.

Human prion diseases are fatal neurodegenerative disorders characterized by neuronal damage in brain. Protein S-nitrosylation, the covalent adduction of a NO to cysteine, plays a role in human brain biology, and brain dysfunction is a prominent feature of prion disease, yet the direct brain targets of S-nitrosylation are largely unknown. We described the first proteomic analysis of global S-nitrosylation in brain tissues of sporadic Creutzfeldt-Jakob disease (sCJD), fatal familial insomnia (FFI), and genetic CJD with a substitution of valine for glycine at codon 114 of the prion protein gene (G114V gCJD) accompanying with normal control with isobaric tags for relative and absolute quantitation (iTRAQ) combined with a nano-HPLC/Q-Exactive mass spectrometry platform. In parallel, we used several approaches to provide quality control for the experimentally defined S-nitrosylated proteins. A total of 1509 S-nitrosylated proteins (SNO-proteins) were identified, and data are available via ProteomeXchange with identifier PXD002813. The cerebellum tissues appeared to contain more commonly differentially expressed SNO-proteins (DESPs) than cortex of sCJD, FFI, and gCJD. Three selected SNO-proteins were verified by Western blots, consistent with proteomics assays. Gene ontology analysis showed that more up-regulated DESPs were involved in metabolism, cell cytoskeleton/structure, and immune system both in the cortex and cerebellum, while more down-regulated ones in both regions were involved in cell cytoskeleton/structure, cell-cell communication, and miscellaneous function protein. Pathway analysis suggested that systemic lupus erythematosus, pathogenic Escherichia coli infection, and extracellular matrix-receptor interaction were the most commonly affected pathways, which were identified from at least two different diseases. Using STRING database, the network of immune system and cell cytoskeleton and structure were commonly identified in the context of the up-regulated and down-regulated DESPs, respectively, both in the cortex and cerebellum. Our study thus have implications for understanding the molecular mechanisms of human prion diseases related to abnormal protein S-nitrosylation and pave the way for future studies focused on potential biomarkers for the diagnosis and therapy of human prion diseases.

Remarkable impairment of Wnt/ß-catenin signaling in the brains of the mice infected with scrapie agents.

Prion diseases are a group of neurodegenerative diseases characterized by neuronal loss and spongiform degeneration, astrogliosis and aggregation of scrapie prion protein (PrPSc ) in the central nervous system (CNS). The Wnt signaling pathway is a highly evolutionarily conserved pathway in eukaryotes that regulates cell proliferation, differentiation and survival. Impairment of Wnt/ß-catenin signaling has been reported in the CNS of various neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. To investigate the functional state of Wnt/ß-catenin signaling in the CNS tissues during the progression of prion disease, the components of Wnt/ß-catenin signaling in the brains of the scrapie agents 139A- and ME7-infected mice were evaluated. Compared with the normal controls, the brain levels of phosphor-ß-catenin (Ser33,37 and Thr41 ) in 139A- and ME7-infected mice were significantly increased, while those of cyclin D1, which is one of the target genes of Wnt signaling, were decreased. The levels of phosphor-glycogen synthase kinase-3ß (GSK-3ß) Ser9 were markedly reduced, representing an enhanced GSK-3ß activity in scrapie-infected mice. Both western blot and immunohistochemical assays revealed a remarkable increase of Dickkopf-1, the antagonist of Wnt/ß-catenin signaling, in the brains of scrapie-infected anim-als, which co-localized well with the remaining neurons in the immunofluorescent tests. We also observed slightly decreased Wnt-3 and unchanged disheveled-3 (Dvl-3) in the brains of the infected mice. Our data, here, strongly indicate an impairment of Wnt/ß-catenin pathway in the brains of prion disease, which shows a time-dependent progression along with the incubation period. Schematic for the impairment of canonical Wnt signaling during prion infection. The left and right parts represent the normal and prion-infected situations, respectively. Prion infection or PrPSc accumulation triggers the over-expression of Dickkopf WNT signaling pathway inhibitor 1 (DKK-1) and the enhancement of glycogen synthase kinase 3ß (GSK-3ß) activity, which subsequently promotes the phosphorylation and degradation of ß-catenin. As a result, the impairment of ß-catenin signaling leads to the down-regulation of Wnt target genes.

The Levels of Tau Isoforms Containing Exon-2 and Exon-10 Segments Increased in the Cerebrospinal Fluids of the Patients with Sporadic Creutzfeldt-Jakob Disease.

The alteration of protein tau in the cerebrospinal fluid (CSF) of Creutzfeldt-Jakob disease (CJD) has been widely evaluated, possessing a significant diagnostic value for CJD. With the biotin-labeled tau-exon-specific mAbs, direct ELISA methods were established and the levels of tau isoforms containing exon-2 and exon-10 segments in CSF of the patients with various human prion diseases and in brain tissues of scrapie-infected animals were evaluated. The results showed that the levels of tau, especially containing four repeats in microtubule binding domain, were increased in the CSF samples of the patients with sporadic CJD (sCJD). Using the unlabeled (cold) mixed exon-specific mAbs, a competitive tau ELISA was conducted based on a commercial tau kit. It revealed that the majority of the increased tau in the CSF of sCJD cases was derived from the tau isoforms with exon-2 and exon-10 segments. Increases of CSF tau isoforms with exon-2 and exon-10 segments were also observed in the patients of E200K and T188K genetic CJD (gCJD), but not in the cases of fatal familiar insomnia (FFI). The increasing levels of tau isoforms with exon-2 and exon-10 segments in the group of sCJD correlated well with the positive 14-3-3 in CSF. Additionally, the similar alterative profiles of tau isoforms with exon-2 and exon-10 segments were also observed in the brain tissues of scrapie-infected rodents and a sCJD patient. Our data here propose the tau isoforms with exon-2 and exon-10 segments increase in CSF of sCJD and some types of gCJD, which may help to understand the physiological metabolism and pathological significance of various tau isoforms in the pathogenesis of prion diseases.