ABSTRACT
1Lab. of Pharmacology, Department of Oncology, Biology and Genetics. University of Genova, V.le Benedetto XV. 2, 16132 Genova, Italy, tel/fax +39 010 3538806. 2Center of Excellence for Biomedical Research (CEBR), University of Genova, e-mail tullio.florio@unige.it.
To define the mechanisms by which hPrP90-231 induces cell death, we analysed its interaction with living cells and monitored its intracellular fate. Treatment of SH-SY5Y cells with fluorescein-5-isotyocianate (FITC)-conjugated hPrP90-231 caused the accumulation of cytosolic aggregates of the prion protein fragment that increased in number and size in a time-dependent manner. The formation of large intracellular hPrP90-231 aggregates correlated with the activation of apoptosis. hPrP90-231 aggregates occurred within lysotracker-positive vesicles and induced the formation of activated cathepsin D, indicating that hPrP90-231 is partitioned into the endosomal-lysosomal system structures, activating the proteolytic machinery. Remarkably, the inhibition of cathepsin D activity significantly reduced hPrP-90-231-dependent apoptosis. Internalised hPrP90-231 forms detergent-insoluble and SDS-stable aggregates, displaying partial resistance to proteolysis. By confocal microscopy analysis of lucifer yellow (LY) intracellular partition, we show that hPrP90-231 accumulation induces lysosome destabilization and loss of lysosomal membrane impermeability. In fact, while control cells evidenced a vesicular pattern of LY fluorescence (index of healthy lysosomes), hPrP90-231-treated cells showed diffuse cytosolic fluorescence, indicating LY diffusion through damaged lysosomes. In conclusion, these data indicate that exogenously-added hPrP90-231 forms intralysosomal deposits having features of insoluble, protease resistant aggregates and could trigger a lysosome-mediated apoptosis by inducing lysosome membrane permeabilization followed by the release of hydrolytic enzymes. Fundings by PRIN 2008 and Compagnia di San Paolo to T.F. are gratefully acknowledged.