Abnormal activation of microglia accompanied with disrupted CX3CR1/CX3CL1 pathway in the brains of the hamsters infected with scrapie agent 263K.

Microglial cells are resident mononuclear phagocytes of the central nervous system (CNS). Active proliferation of microglia in the brain has been identified in neurodegenerative disorders, including some kinds of prion disease. However, the detailed regional distribution between microglia and PrP(Sc) deposition has not been presented, and investigation of fractalkine signaling which is involved in the regulation of activation of microglia in prion disease is not well documented. In this study, the disease phenomenon of microglial accumulation in the CNS was thoroughly analyzed using a scrapie-infected experimental model. Western blots of microglia-specific markers Iba1 and CD68, immunohistochemical and immunofluorescent assays demonstrated obviously activation of microglia in almost whole brain regions in the infected animals. Under the dynamic analysis on hallmarks of activation of microglia, a time-dependent increase of Iba1 and CD68 was detected, accompanied by accumulation of PrP(Sc) and progression of neurodegenerative symptoms. With serial brain sections and double staining of Iba1 and PrP(Sc), we observed that the microglia distributed around PrP(Sc) deposits in 263K-infected hamsters' brains, proposing PrP(Sc) phagocytosis. Flow cytometry assays with the single-cell suspensions prepared from the cortical region of the infected brains verified an activation of microglial population. ELISA assays of the cytokines in brain homogenates revealed significant upregulations of interleukin (IL)-1ß, IL-6 and TNF-α when infected. Evaluation of fractalkine signaling in the infected hamsters' brains showed progressively downregulation of CX3CL1 during the incubation. Prion peptide PrP106-126 also disrupted fractalkine and evoked microglial activation in rat primary neuron-glia mixed cultures. Our data here demonstrate an activated status of microglia in CNS tissues of infectious prion disease, possibly through fractalkine signaling deficiency.

Global transcriptional profiling of the postmortem brain of a patient with G114V genetic Creutzfeldt-Jakob disease.

Familial or genetic Creutzfeldt-Jakob disease (fCJD or gCJD) is an inherent human prion disease caused by mutation of the prion protein gene (PRNP). In the present study, global expression patterns of the parietal cortex from a patient with G114V gCJD were analyzed using the Affymetrix Human Genome U133+ 2.0 chip with a commercial normal human parietal cortex RNA pool as a normal control. In total, 8,774 genes showed differential expression; among them 2,769 genes were upregulated and 6,005 genes were downregulated. The reliability of the results was confirmed using real-time RT-PCR assays. The most differentially expressed genes (DEGs) were involved in transcription regulation, ion transport, transcription, cell adhesion, and signal transduction. The genes associated with gliosis were upregulated and the genes marked for neurons were downregulated, while the transcription of the PRNP gene remained unaltered. A total of 169 different pathways exhibited significant changes in the brain of G114V gCJD. The most significantly regulated pathways included Alzheimer's and Parkinson's disease, oxidative phosphorylation, regulation of actin cytoskeleton, MAPK signaling and proteasome, which have previously been linked to prion diseases. In addition, we found some pathways that have rarely been explored in regards to prion diseases that were also significantly altered in G114V gCJD, such as axon guidance, gap junction and purine metabolism. The majority of the genes in the 10 most altered pathways were downregulated. The data of the present study provide useful insights into the pathogenesis of G114V gCJD and potential biomarkers for diagnostic and therapeutic purposes.

Comparison of the pathologic and pathogenic features in six different regions of postmortem brains of three patients with fatal familial insomnia.

Fatal familial insomnia (FFI) is an autosomal dominant prion disease clinically characterized by rapidly progressive insomnia, prominent autonomic alterations and behavioral disturbance. The D178N mutation of the prion protein gene (PRNP) on chromosome 20 in conjunction with methionine at codon 129 is a molecular feature. Although the neuropathological characteristics of FFI are well documented, the neuropathologic and pathogenic features of FFI patients remain poorly understood. Six brain regions of postmortem brains from 3 FFI patients were examined using immunohistochemistry, western blot analyses and quantitative real-time PCR. In all 3 brain specimens, reactive astrogliosis was found to be more severe in the thalamus than in the cortex regions. Western blot analyses showed that all three brains expressed PrP, but only 2 were associated with significantly weak proteinase K (PK) resistance. However, the conformational stabilities of PrPSc in the 3 FFI brains were significantly weaker than those presented in a G114V genetic Creutzfeldt-Jakob disease (gCJD) case. Immunohistochemistry and western blot analyses showed comparable amounts of neuron-specific enolase (NSE)-positive stained cells and NSE protein among the different regions in the three brains. In addition, the transcriptional levels of glial fibrillary acidic protein (GFAP) and NSE-specific mRNAs were coincident with the expression of these proteins. In conclusion, in the present study, we described the detailed regional neuropathology of FFI cases.

Reduction of protein kinase MARK4 in the brains of experimental scrapie rodents and human prion disease correlates with deposits of PrP(Sc).

Microtubule affinity-regulating kinase 4 (MARK4) belongs to a family of kinases that are able to actively phosphorylate the neuronal microtubule-associate proteins (MAPs), such as tau, MAP2 and the ubiquitous MAP4. Abnormal changes in tubulin and the profiles of tau have been previously reported in the human brain and animal transmissible spongiform encephalopathies (TSEs), which may be associated with abnormal alterations of various cellular kinases. To elucidate the possible role of MARK4 in TSE pathogenesis, the MARK4 levels in the brain tissues of scrapie-infected rodents and human prion diseases were evaluated using western blotting and immunohistochemical assays. The results revealed that at terminal stages of the diseases, MARK4 levels in the brain tissues of the scrapie 263K-infected hamsters, 139A-infected mice and a case of Creutzfeldt-Jakob disease (CJD, G114V gCJD) correlated with amounts of PrP(Sc) deposits that were almost undetectable. On the other hand MARK4 signals were noticeable in the brain tissues of a fatal familial insomnia (FFI) patient without PrP(Sc). The reduction of MARK4 was closely related to the prolonged incubation times. These results could be reproduced in SK-N-SH and PC12 cell lines after being exposed to the synthetic peptide PrP106-126. Accordingly, the levels of phosphorylated tau at Ser262 (p-tau262) in cultured cells exposed to PrP106-126, or the ratios of p-tau262/total tau in the brain tissues of 263K-infected hamsters were also significantly decreased. According to our data there is a correlation between a TSE pathological-associated decline of MARK4 in the brain tissues with the deposits of PrP(Sc). Reduction of MARK4 will result in abnormalities of tau phosphorylation, and possibly induce further detachment of microtubules and hinder microtubule transportation.

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.

Molecular interaction of TPPP with PrP antagonized the CytoPrP-induced disruption of microtubule structures and cytotoxicity.

BACKGROUND: Tubulin polymerization promoting protein/p25 (TPPP/p25), known as a microtubule-associated protein (MAP), is a brain-specific unstructured protein with a physiological function of stabilizing cellular microtubular ultrastructures. Whether TPPP involves in the normal functions of PrP or the pathogenesis of prion disease remains unknown. Here, we proposed the data that TPPP formed molecular complex with PrP. We also investigated its influence on the aggregation of PrP and fibrillization of PrP106-126 in vitro, its antagonization against the disruption of microtubule structures and cytotoxicity of cytosolic PrP in cells, and its alternation in the brains of scrapie-infected experimental hamsters. METHODOLOGY/PRINCIPAL FINDINGS: Using pull-down and immunoprecipitation assays, distinct molecular interaction between TPPP and PrP were identified and the segment of TPPP spanning residues 100-219 and the segment of PrP spanning residues 106-126 were mapped as the regions responsible for protein interaction. Sedimentation experiments found that TPPP increased the aggregation of full-length recombinant PrP (PrP23-231) in vitro. Transmission electron microscopy and Thioflavin T (ThT) assays showed that TPPP enhanced fibril formation of synthetic peptide PrP106-126 in vitro. Expression of TPPP in the cultured cells did not obviously change the microtubule networks observed by a tubulin-specific immunofluorescent assay and cell growth features measured by CCK8 tests, but significantly antagonized the disruption of microtubule structures and rescued the cytotoxicity caused by the accumulation of cytosolic PrP (CytoPrP). Furthermore, Western blots identified that the levels of the endogenous TPPP in the brains of scrapie-infected experimental hamsters were significantly reduced. CONCLUSION/SIGNIFICANCE: Those data highlight TPPP may work as a protective factor for cells against the damage effects of the accumulation of abnormal forms of PrPs, besides its function as an agent for dynamic stabilization of microtubular ultrastructures.

Knockdown of prion protein (PrP) by RNA interference weakens the protective activity of wild-type PrP against copper ion and antagonizes the cytotoxicity of fCJD-associated PrP mutants in cultured cells.

Development of the pathogenesis of transmissible spongiform encephalopathies (TSEs) requires the presence of both the normal host prion protein (PrPC) and the abnormal pathological proteinase-K resistant isoform (PrPSc). Reduction of PrPC levels has been shown to extend survival time after prion infection. In this report, based on analysis of the known sequences of human PrP, we constructed two small interfering RNA (siRNA) duplexes targeting the segments of amino acids (aa) 108-114 (Ri2) and aa 171-177 (Ri3). Western blot analysis results revealed that these PrP-specific siRNAs could effectively knock down the levels of either endogenous PrP in human neuroblastoma SHSY-5Y cells or recombinant PrP transfected with the plasmid expressing the full-length human PrP in human embryonic kidney (HEK) 293T cells. Meanwhile, the two siRNAs also showed a significant effect on the reduction of the expression of the PrP-PG9 and PrP-PG12 familial Creutzfeldt-Jakob disease (CJD)-associated PrP mutants with four and seven extra octarepeats, in the cells transfected with the respective expression plasmids. MTT tests identified that knockdown of wild-type PrP by Ri2 and Ri3 did not change the cell growth capacities, but significantly decreased the cell resistances against the challenge of Cu2+. Co-expression of Ri2 and Ri3 partially antagonized the cytotoxicity caused by expressing PrP-PG9 and PrP-PG12 in the two cell lines. Moreover, the rescuing effectiveness of PrP siRNAs was time-related, with the more efficient antagonism of the cytotoxicity of fCJD-associated PrP mutants occurring at the early stages after transfection. The data shown here provide useful clues for seeking potential therapeutic tools for prion diseases.

A novel PrP partner HS-1 associated protein X-1 (HAX-1) protected the cultured cells against the challenge of H2O2.

Prion protein (PrP) is a ubiquitous conserved glycoprotein predominantly expressed in neurons of the central nervous system (CNS). To elucidate on its cellular function, we performed a yeast two-hybrid screen within an adult human brain cDNA library for potential PrP-binding molecules. A novel protein, HS-1 associated protein X-1 (HAX-1), was identified to be able to bind with PrP strongly. The interaction between the two proteins has been further verified by glutathione-S-transferase (GST) pull-down and immunoprecipitation assays. The minimal binding regions were mapped to the segments of residues aa 91-163 for PrP(C) and residues aa 38-129 for HAX-1. Immunofluorescent assays of co-expressions of human PrP and HAX-1 in 293T and SHSY-5Y cells revealed marked co-localizations of those two proteins in cytoplasm. Moreover, the co-expression of HAX-1 and wild-type PrP (PG5) was found to enhance the cellular resistance to the challenge of H2O2. Contrarily, co-transfection of HAX-1 did not reverse but aggravated the cytotoxicities of the genetic CJD (gCJD) associated PrP mutants with nine- (PG9) and fourteen-octarepeats (PG14). Our data provide for the first time a new PrP-interacting partner that may play role in cell oxidative stress and anti-apoptosis physiologically and cell damage pathologically.

Establishment of hamster- and human-PRNP transgenic mice.

OBJECTIVE: To create transgenic mice expressing hamster- and human-PRNP as a model for understanding the physiological function and pathology of prion protein (PrP), as well as the mechanism of cross-species transmission of transmissible spongiform encephalopathies (TSEs). METHODS: Hamster and human-PRNP transgenic mice were established by conventional methods. The copy number of integrated PRNP in various mouse lines was mapped by real-time PCR. PRNP mRNA and protein levels were determined by semi-quantitative RT-PCR, real-time RT-PCR, and western blot analysis. Histological analyses of transgenic mice were performed by hematoxylin and eosin (H & E) staining and immunohistochemical (IHC) methods. RESULTS: Integrated PRNP copy number in various mouse lines was 53 (Tg-haPrP1), 18 (Tg-huPrP1), 3 (Tg-huPrP2), and 16 (Tg-huPrP5), respectively. Exogenous PrPs were expressed at both the transcriptional and translational level. Histological assays did not detect any abnormalities in brain or other organs. CONCLUSION: We have established one hamster-PRNP transgenic mouse line and three human-PRNP transgenic mouse lines. These four transgenic mouse lines provide ideal models for additional research.

[The enhancement of DNA binding ability of a mutated E2 (A338V) protein of HPV-2].

HPV-2 is a very common type of HPV which causes common warts. The E2 protein of virus can repress the activity of the viral early promoter through binding to the specific binding sites in viral LCR. Previously we reported that the repression of a mutated E2 protein of HPV-2 isolated from a patient with huge common wart on the viral early promoter was obviously decreased, and A338V mutation located at the C terminal DNA binding region of E2 protein. In this study, we expressed and purified the recombinant mutated and prototype E2 fusion proteins, both in the contexts of the C terminal and the full length, by prokaryotic expression system. The electrophoretic mobility shift assay showed E2 protein could bind to double-stranded DNA oligos labeled with biotin that covered two E2 binding sites. The DNA binding abilities of both C terminal and full-length mutated E2 proteins were stronger than the prototype analogs. This result indicates that the enhancement of the mutated E2 DNA binding ability may be the molecular mechanism for its impact on the activity of viral promoter, which correlates with the phenotype of extensive common wart.