title: Prion protein with Y145STOP mutation induces mitochondria-mediated apoptosis and PrP-containing deposits in vitro.
abstract: A pathogenic truncation of an amber mutation at codon 145 (Y145STOP) in Gerstmann-Straussler-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.
authors.affiliation: Department of Cortical Function Disorders, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan.
authors.forename: Naomi S
authors.initials: NS
authors.lastname: Hachiya
authors.forename: Kota
authors.initials: K
authors.lastname: Watanabe
authors.forename: Makiko Y
authors.initials: MY
authors.lastname: Kawabata
authors.forename: Akiko
authors.initials: A
authors.lastname: Jozuka
authors.forename: Yoshimichi
authors.initials: Y
authors.lastname: Kozuka
authors.forename: Yuji
authors.initials: Y
authors.lastname: Sakasegawa
authors.forename: Kiyotoshi
authors.initials: K
authors.lastname: Kaneko
mesh_terms: D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D003062:Codon / Q000235:genetics*; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D016098:Gerstmann-Straussler-Scheinker Disease / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000502:physiology*; D009154:Mutation; D009447:Neuroblastoma / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology; D011328:Prions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*
chemical_list: D003062:Codon; D018696:Neuroprotective Agents; D011328:Prions; D045304:Cytochromes c; D004247:DNA