In-situ analysis of cellular poly(ADP-ribose) production in scrapie-infected mouse neuroblastoma cells.
Transmissible spongiform encephalopathies (TSEs), also called prion diseases, are characterized by formation of the disease-associated isoform of prion protein (PrP(Sc)), which arises from a normal isoform termed PrP(c) by a post-translational conversion process occurring in an autocatalytic fashion. Oxidative stress has been proposed as a pathogenetic mechanism in TSEs and increased lipid peroxidation has recently been described in prion-infected cell cultures, suggesting an intrinsic link between the presence of prions and oxidative stress. We investigated if poly(ADP-ribose) formation can be detected in cultured cells upon prion infection, as this NAD+-consuming and DNA strand break-activated nuclear enzymatic reaction has the potential to cause rapid and lethal NAD+ depletion in cells under severe oxidative stress. Poly(ADP-ribose) production was analysed by immunofluorescence in freshly scrapie-infected Neuro2a-D11 mouse neuroblastoma cells, which had been confirmed by immunocytochemistry to produce PrP(Sc), and in uninfected controls. No spontaneous poly(ADP-ribose) specific signals were observed in infected or in uninfected cells, while both cell types readily reacted to H2O2 treatment with poly(ADP-ribose) synthesis in a dose-dependent manner, with no obvious difference in staining intensity at any dose tested. In summary, our data reveal that replication of scrapie agent in neuroblastoma cells can proceed without detectable stimulation of the cellular poly(ADP-ribosyl)ation system.
Zhang
Yonghua
Y
School of Clinical Medical Sciences-Gerontology, Institute for Ageing and Health, University of Newcastle upon Tyne, Wolfson Research Centre, NGH, Westgate Road, Newcastle-upon-Tyne NE4 6BE, UK.
Poirier
Guy G
GG
Bürkle
Alexander
A
D000818:Animals; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis*; D018620:PrPSc Proteins / Q000378:metabolism* / Q000472:pathogenicity; D011328:Prions / Q000472:pathogenicity; D012608:Scrapie / Q000635:transmission*; D014407:Tumor Cells, Cultured
D018620:PrPSc Proteins; D011328:Prions; D011064:Poly Adenosine Diphosphate Ribose; D006861:Hydrogen Peroxide