FORPRION

BAVARIAN RESEARCH ASSOCIATION PRIONS

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TUM 7 The Role of Activated Retroviral Genes as Cofactors in Prion-induced Spongiform Encephalopathy: Studies with a Mouse Model for Pathogenesis and Therapy


http://www.gsf.de/tum.html

Prions as causative agents of transmissible spongiform encephalopathies have been well-investigated in experimental and modelling work. However, little is known about the molecular pathogenesis of prion-induced encephalopathies, the role of co-factors and the interaction of prions with cellular components. In this project, we used different microarray approaches to identify genes that change regulation in response to prion infection. First, we investigated the influence of prion infection on expression of murine endogenous retroviruses (ERVs), which compose approximately 10% of the mouse genome. Activation of endogenous retroviruses could result in the production of retroviral particles, which may serve as vehicles for spreading pathogenic prion proteins to other cells. Hypothalamic neuronal cells (GT1) and neuroblastoma cells (N2a) can be persistently infected with mouse adapted scrapie strains. Using a retrovirus-specific DNA microarray and quantitative PCR methods, we compared the expression profiles of ERVs in prion-infected, uninfected and anti-prion compound treated murine neuronal cell lines, including clonal cell populations. The results indicate that prion infection influences ERV expression in neuronal cell lines, that this influence is cell line-specific, ERV-specific, and is responsive to anti-prion compound treatment. In particular, murine leukemia viruses (MLV) and retroviral elements encoding intracisternal A particles (IAP-1) were found to be consistently upregulated in prion-infected N2A cells. These findings were supported by experiments of other groups reporting that coinfection with exogenous moloney murine leukaemia virus (MoMLV) strongly enhances the release of scrapie infectivity in the supernatant of prion-infected cells. Taken together, these data suggest that prions may not only be spread by retoviral particles but moreover may stimulate the production of such virions. The human genome contains about the same amount of retroviral sequences as the mouse genome. Although these sequences are by far less active than murine endogenous retroviruses, at least some of them are able to produce retrovirus-like particles or express retroviral Env proteins that could mediate cell to cell contact and thus contribute to prion spreading. In a second approach, we used GT1 and N2a cells, as well as an N2a cell clone that was newly infected with either the mouse adapted prion strain 22L or exposed to uninfected brain homogenate as a negative control, to identify cellular mouse gene influenced by prion infection. Large-scale expression analysis was performed exploiting a mouse cDNA microarray comprising about 21,000 spotted sequences of expressed mouse genes. Real-Time PCR of selected genes was used for confirmation of the microarray data and for semi-quantitative analysis. Several hundred genes were identified that are differentially expressed in the different prion-infected cell lines when compared to uninfected cells. Prion-infected N2a and GT1 cells demonstrate unique changes in RNA profiles and both differ from the reported changes in human microglia and prion-infected brain studies albeit with some overlap. Furthermore, several of the identified changes in gene expression have also been reported for other neurodegenerative diseases such as Alzheimer’s disease. The results illustrate that prion infection differs in effect depending on cell type, which could be exploited for diagnostic or therapeutic intervention. 

Information

Launching date

07.2001

End

06.2007

Funded by

Bavarian State Ministry for Environmental Affairs and Consumer Protection