Characterization of MPP+-induced cell death in a dopaminergic neuronal cell line: Role of macromolecule synthesis, cytosolic calcium, caspase, and Bcl-2-related proteins

To further characterize MPP+-induced cell death and to explore the role of Bcl-2-related proteins in this death paradigm, we utilized a mesencephalon- derived dopaminergic neuronal cell line (MN9D) stably transfected with huma n bcl-2 (MN9D/Bcl-2), its C-terminal deletion mutant (MN9D/Bcl-2 Delta 22), murine bar (MN9D/Bax), or a control vector (MN9D/Neo). As determined by el ectron microscopy and TUNEL assay, MN9D/Neo cells exposed to MPP+ underwent a cell death that was characterized by mitochondrial swelling and irregula rly scattered heterochromatin without accompanying DNA fragmentation. Howev er, cell swelling typically seen in necrosis did not appear. To examine the biochemical events associated with MPP+-induced cell death, various analys es were conducted. Addition of a broad-spectrum caspase inhibitor, N-benzyl oxycarbonyl-Val-Ala-Asp-fluoromethylketone (50-400 mu M) or Boc-aspartyl(OM e)-fluoromethylketone (50-200 mu M) did not attenuate MPP+-induced cell dea th while the same treatment protected MN9D/Neo cells against staurosporine- induced apoptotic cell death. Concurrent treatment with an inhibitor of mac romolecule synthesis such as cycloheximide, emetine, or actinomycin D block ed MPP+-induced cell death, suggesting that new protein synthesis is requir ed as demonstrated in many apoptotic cell death. The level of cytosolic cal cium in MN9D/Neo cells was unchanged over 24 h following MPP+ treatment, as monitored by means of the fluorescent probe Fura-8. Western blot analysis indicated that expression level of proapoptotic protein, Bar was not signif icantly altered after MPP+ treatment. In this death paradigm, overexpressio n of Bcl-2 but not its C-terminal deletion mutant attenuated MPP+-induced c ell death whereas overexpression of Bar had no effect. Taken together, thes e data indicate that (i) MPP+ induces a distinct form of cell death which r esembles both apoptosis and necrosis; and (ii) full-length Bcl-2 counters M PP+-induced morphological changes and cell death via a mechanism that is de pendent on de novo protein synthesis but independent of cytosolic calcium c hanges, fax expression, and/or activation of caspase(s) in MN9D cells. (C) 1999 Academic Press.