NR AREB
AU Kretlow,A.; Wang,Q.; Beekes,M.; Miller,L.; Naumann,D.
TI In situ characterization of prion protein structure and metal accumulation in scrapie-infected cells by synchrotron infrared and x-ray microspectroscopy
QU TSE-Forum, 4. Kongress - Nationale TSE-Forschungsplattform, Düsseldorf 28.10.-29.10.2004, Poster GL-10
PT Konferenz-Poster
AB
Transmissible spongiform encephalopathies (TSEs) such as scrapie are neurodegenerative diseases that are characterized by the accumulation of a misfolded prion protein. During the pathogenesis, the normally alpha-helical prion protein (PrPc) refolds to the partially proteinase K-resistant abnormal form (PrPsc), which is high in ß-sheet content. In this study, dorsal root ganglia from the 263K hamster model of scrapie were cut into 8 µm thin sections and mounted onto a metal-free glass slide coated with aluminum. This substrate was chosen because it is reflective to infrared light and transparent to hard x-ray radiation, permitting both, synchrotron infrared and x-ray microspectroscopies to be performed on the same sample. Single neuronal cells were mapped with synchrotron infrared microspectroscopy to study protein structure and x-ray fluorescence microprobe to determine metal accumulation. Both techniques provided a spatial resolution of approximately 6 microns. Results showed that some IR spectra of the infected cells exhibited a significant frequency shift of the Amide I band, where an increased peak intensity in the 2nd derivative spectra was observed at ~1634 cm-1 (attributed to beta sheet) and a decreased peak intensity at ~1659 cm-1 was also seen (attributed to alpha helix). The investigated ganglia were also stained with an antibody for the prion protein in order to confirm that the IR observations were associated with accumulated PrPsc. Finally, the same cells were also measured by x-ray microspectroscopy for the first time. Preliminary results showed an accumulation of Zn and Cu in cells infected with PrPsc.
This work is supported by the National Institutes of Health Grant R01-GM66873. The NSLS is supported by the United States Department of Energy under contract DE-AC02-98CH10886.
Figure 1. Infrared micro-spectra of two neuronal cells. (A) Frequency map overlapped with corresponding tissue section; (B) Infrared micro-spectra of areas with (red) and without (blue) PrPsc accumulation; (C) Second derivatives of the spectra shown in (B).
AD Ariane Kretlow, Dieter Naumann, P 25, Robert Koch-Institut Berlin, Nordufer 20, 13353 Berlin; Qi Wang, Lisa Miller, National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY11973; Michael Beekes, P 24, Robert Koch-Institut Berlin, Nordufer 20, 13353 Berlin
SP englisch
PO Deutschland
OR Tagungsband