FORPRION

BAVARIAN RESEARCH ASSOCIATION PRIONS

LMU 9 Phenotyping of Prion Diseases using GFP-PrP Chimeras in Transgenic Mice

The immediate cellular processes and pathogenetic mechanisms leading to prion diseases following infection are not well understood. A primary aim of this project was to gain insight into these processes by the generation of transgenetic mouse lines expressing modified versions of prion protein (PrP) under the control of authenitic mouse Prnp gene promotor elements and the use these mouse lines in appropriate animal experiments addressing PrP function and pathomechanisms of prion disease. The first type of transgenic mice created for this purpose expresses a chimeric PrP fused to the enhanced green fluorescent protein (EGFP-PrP). We have obtained five distinct lines of transgenic animals carrying this type of transgene. Three of which show the same distribution of the EGFP-PrP fusion protein in their brains as is observed for PrP alone in wildtype animals. Two transgenic lines differ slightly in the observed EGFP-PrP distribution pattern from the normal PrP distribution pattern. All these transgenic lines are now bread on a normal wildtype mouse background as well as on PrP-knockout background. Animals on wildtype background and those on a hemizygous wildtyp/PrP-knockout background could already be inoculated with prions of the RML strain via intracerebral and oral routes. The EGFP-PrP lines on wildtype background had the same scrapie incubation time as normal wildtype animals, when infected intracerebrally. In brain homogenates of terminally ill animals from these EGFP-PrP expressing lines we were able to detect a PK-resistent PrP portion of the fusion protein indicating the conversion of this PrP-variant to the PrPSc conformation. Due to the intrinsic fluorescent label of the protein we are now able to monitor early changes in the localization of the EGFP-PrP fusion protein during infection and identify the first target cells in particular during oral infection of the intestinal tract. Therefore, with these transgenic mouse lines we have now in hand an extremly versatile tool to examine prion pathogenesis and divers aspects of PrP-function under physiological conditions within the intact organism. In the second part of the project we generated and examined transgenic mice carrying PrP mutagenized at histidine residues wihin its copper-binding sites. A second type of transgenic animals expresses PrP with an exchange of all four histidine residues of the octarepeat region to glycines. From the four distinct transgenic lines obtained two, which were bread on a PrP-knockout background, could already be used for infection studies aiming to clarify the potential role of the interaction of copper and PrPC in the infectious process. Line HG34 had a dramatic prolongation of the scrapie survival time, which was more than doubled, when infected with the RML strain, as compared to wildtype animals. Also the HG1 line showed a significant prolongation of scrapie survial times although less dramatic. The mutant PrP produced by these lines is fully functional as it can the completely suppress the neurodegeneration of the “Shmerling”-phenotype of transgenic F35 animals that express a truncated PrP-version. The results of in vitro aggregation assays with bacterially expressed HG-PrP as well as differences observed in the proteolytic cleavage pattern of PrP in brain homogenate of scrapie infected HG34-mice as compared to wildtype point to a possible significant influence of copper-binding (or the copper-binding sites) on the pathogenesis of prion disease. The absence of intact copper-binding sites within the octapeptide repeat region of PrP seems slow down disease progression in vivo and aggregation in vitro quite severely. In order to clarify the role of copper-binding sites outside the octarepeat, we have generated a third type of transgenic mice which expresses PrP with only histindine 96 exchanged to glycine. Of the five transgenic lines obtained for this construct one line has already been bread to PrP-knockout background. Infection of such transgenic animals with RML scrapie has lead to the astonishing observation of a dramatically reduced survival time. Thus raising the possibility that specific copper-binding sites within PrP have largely different roles in the disease progress.