Chronic exposure to sub-lethal beta-amyloid (Abeta) inhibits the import of nuclear-encoded proteins to mitochondria in differentiated PC12 cells.

Studies on amyloid beta (Abeta|), the peptide thought to play a crucial role in the pathogenesis of Alzheimer's disease, have implicated mitochondria in Abeta-mediated neurotoxicity. We used differentiated PC12 cells stably transfected with an inducible green fluorescent protein (GFP) fusion protein containing an N'-terminal mitochondrial targeting sequence (mtGFP), to examine the effects of sub-lethal Abeta on the import of nuclear-encoded proteins to mitochondria. Exposure to sub-lethal Abeta(25-35) (10 mumol/L) for 48 h inhibited mtGFP import to mitochondria; average rates decreased by 20 +/- 4%.

Flow cytometry and GFP: a novel assay for measuring the import and turnover of nuclear-encoded mitochondrial proteins in live PC12 cells.

Background: Mitochondrial protein import is typically measured by adding radiolabeled precursor proteins to isolated mitochondria. We have developed a novel, high-throughput method for measuring protein import in live differentiated PC12 cells using a tetracycline (Tet) regulated, nuclear encoded, mitochondrially-targeted GFP fusion protein and flow cytometry.

Methods: We generated a PC12 cell line stably transfected with an inducible GFP fusion protein (GFPmt) targeted to mitochondria.