NR AWOV
AU Thellung,S.; Corsaro,A.; Villa,V.; Florio,T.
TI Sheet-structured PrP90-231 induces SH-SY5Y cell death through intracellular accumulation
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions CE-47
PT Konferenz-Poster
AB Peptides corresponding to amyloidogenic regions of the prion protein showed, at some extent, PrPsc physicochemical features and possess neurotoxicity in vitro. Recently, we have demonstrated that the recombinant peptide corresponding to the 90-231 region of human prion protein (PrP90-231) triggers SH-SY5Y human neuroblastoma cell death, once refolded into a ß-sheet-rich structure. To follow PrP90-231 interaction with cells in relation with its toxicity, we have conjugated PrP90-231 to the fluorescent molecule fluorescein-5-isothiocyanate (FITC). By immunoblotting and MTT reduction test, we have observed that PrP90-231 does not undergo proteolytic cleavage due to the tagging process and does not loose its citotoxicity in vitro. Hence we have treated SH-SY5Y cells with subtoxic and toxic concentrations of PrP90-231-FITC (10 nM and 1 M respectively) to analyse, by live cell confocal imaging, the interaction of both peptide concentrations with cells. By using organelle-specific viable trackers, we observed that, after 12-24 hours of treatment at both concentrations, PrP90-231FITC accumulated in SH-SY5Y cytoplasm and localised into organelles showing endo-lysosomal features. At treatment times longer than 48 hours, cells treated with PrP90-231-FITC 10 nM showed the coalescence of peptide-containing bodies into a perinuclear area without evidencing signs of cell degeneration. Conversely, cells treated with 1 M showed a widespread distribution of large bodies containing PrP90-231-FITC and evidenced features of cell death. Moreover, we have measured, by immunoblotting, PrP immunoreactivity in SH-SY5Y lysates after cell treatment with PrP90-231-FITC 1 M (exposure times ranging from 30 minutes to 48 hours). We have observed that, after 120 minutes, a 16 KDa immunoreactive band was detected indicating binding/internalisation of full-length peptide. After 24 hours, full-length peptide immunoreactivity decreased and lower molecular bands appeared. In conclusion, our data demonstrate that PrP90-231 is internalised and subjected to partial proteolysis by SH-SY5Y; we hypothesize that cell death process, in vitro, is triggered when the amount of internalised peptide saturates cellular proteolytic capacity (Grants by PRIN 2004 to TF).
AD Sect. of Pharmacology. Dept. of Oncology, Biology and Genetics. Univ. of Genova, Italy. E-mail: thellung@yahoo.com
SP englisch
PO Italien