NR ARPT
AU Hachiya,N.S.; Watanabe,K.; Kawabata,M.Y.; Jozuka,A.; Kozuka,Y.; Sakasegawa,Y.; Kaneko,K.
TI Prion protein with Y145STOP mutation induces mitochondria-mediated apoptosis and PrP-containing deposits in vitro
QU Biochemical and Biophysical Research Communications 2005 Feb 18; 327(3): 894-9
PT journal article
AB A pathogenic truncation of an amber mutation at codon 145 (Y145STOP) in Gerstmann-Sträussler-Scheinker disease (GSS) was investigated through the real-time imaging in living cells, by utilizing GFP-PrP constructs. GFP-PrP(1-144) exhibited an aberrant localization to mitochondria in mouse neuroblastoma neuro2a (N2a) and HpL3-4 cells, a hippocampal cell line established from prnp gene-ablated mice, whereas full-length GFP-PrP did not. The aberrant mitochondrial localization was also confirmed by Western blot analysis. Since GFP-PrP(1-121), as previously reported, and full-length GFP-PrP do not exhibit such mitochondrial localization, the mitochondrial localization of GFP-PrP(1-144) requires not only PrP residues 121-144 (in human sequence) but also COOH-terminal truncation in the current experimental condition. Subsequently, the GFP-PrP(1-144) induced a change in the mitochondrial innermembrane potential (DeltaPsi(m)), release of cytochrome c from the intermembrane space into the cytosol, and DNA fragmentation in these cells. Non-fluorescent PrP(1-144) also induced the DNA fragmentation in N2a and HpL3-4 cells after the proteasomal inhibition. These data may provide clues as to the molecular mechanism of the neurotoxic property of Y145STOP mutation. Furthermore, immunoelectron microscopy revealed numerous electron-dense deposits in mitochondria clusters of GFP-PrP(1-144)-transfected N2a cells, whereas no deposit was detected in the cells transfected with full-length GFP-PrP. Co-localization of GFP/PrP-immunogold particles with porin-immunogold particles as a mitochondrial marker was observed in such electron-dense vesicular foci, resembling those found in autophagic vacuoles forming secondary lysosomes. Whether such electron-dense deposits may serve as a seed for the growth of amyloid plaques, a characteristic feature of GSS with Y145STOP, awaits further investigations.
MH Animals; *Apoptosis; Blotting, Western; Codon/*genetics; Cytochromes c/metabolism; Cytosol/metabolism; DNA/genetics/metabolism; Gerstmann-Sträussler-Scheinker Disease/genetics/metabolism; Lysosomes/metabolism; Mice; Mitochondria/*physiology; Mutation; Neuroblastoma/metabolism; Neurons/cytology/metabolism; Neuroprotective Agents/pharmacology; Prions/chemistry/genetics/*metabolism/*physiology; Research Support, Non-U.S. Gov't
AD Department of Cortical Function Disorders, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan.
SP englisch
PO USA