Poly(ADP-ribose) immunostaining to detect apoptosis induced by a neurotoxic fragment of prion protein

PrP106-126 is a synthetic peptide representing codons 106-126 of the prion protein, which spontaneously forms amyloid fibrils and exerts neurotoxic ef fects on primary mouse brain cell cultures. Neurotoxicity by this peptide i s commonly used as a model for the neurotoxicity observed in prion diseases and involves the formation of reactive oxygen species which, in turn, can cause DNA damage, including DNA strand breaks. Strand breaks in nuclear DNA can activate poly(ADP-ribose) polymerase to covalently modify nuclear prot eins with poly(ADP-ribose). We, therefore, examined by immunofluorescence w hether or not PrP106-126 triggers poly(ADP-ribose) formation. We observed s trong poly(ADP-ribose) immunofluorescence signals in a fraction of cells, t ypically arranged in a clustered pattern, by 30-48 h after peptide addition . A few positive cells were also present in untreated cultures. Cell morpho logy was suggestive of apoptosis, and this was confirmed by positivity in t he terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (T UNEL) assay. On the other hand, our immunofluorescence assay did not detect any 'early' activation of poly(ADP-ribose) polymerase in morphologically n ormal cells that could have resulted from peptide-induced formation of reac tive oxygen species. We conclude that poly(ADP-ribose) immunostaining is a convenient and reliable method for visualizing cells undergoing apoptosis i nduced by PrP106-126.