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

Won Seok Choi, L. M.T. Canzoniero, S. L. Sensi, Karen L. O'Malley, Byung J. Gwag, Seonghyang Sohn, Ji Eun Kim, Tae H. Oh, Eunhee B. Lee, Young Jun Oh

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Abstract

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 human bcl-2 (MN9D/Bcl-2), its C-terminal deletion mutant (MN9D/Bcl-2Δ22), murine bax (MN9D/Bax), or a control vector (MN9D/Neo). As determined by electron microscopy and TUNEL assay, MN9D/Neo cells exposed to MPP+ underwent a cell death that was characterized by mitochondrial swelling and irregularly scattered heterochromatin without accompanying DNA fragmentation. However, cell swelling typically seen in necrosis did not appear. To examine the biochemical events associated with MPP+-induced cell death, various analyses were conducted. Addition of a broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (50-400 μM) or Boc-aspartyl(OMe)-fluoromethylketone (50-200 μM) did not attenuate MPP+- induced cell death while the same treatment protected MN9D/Neo cells against staurosporine-induced apoptotic cell death. Concurrent treatment with an inhibitor of macromolecule synthesis such as cycloheximide, emetine, or actinomycin D blocked MPP+-induced cell death, suggesting that new protein synthesis is required as demonstrated in many apoptotic cell death. The level of cytosolic calcium in MN9D/Neo cells was unchanged over 24 h following MPP+ treatment, as monitored by means of the fluorescent probe Fura-2. Western blot analysis indicated that expression level of proapoptotic protein, Bax was not significantly altered after MPP+ treatment. In this death paradigm, overexpression of Bcl-2 but not its C-terminal deletion mutant attenuated MPP+-induced cell death whereas overexpression of Bax had no effect. Taken together, these data indicate that (i) MPP+ induces a distinct form of cell death which resembles both apoptosis and necrosis; and (ii) full-length Bcl-2 counters MPP+-induced morphological changes and cell death via a mechanism that is dependent on de novo protein synthesis but independent of cytosolic calcium changes, Bax expression, and/or activation of caspase(s) in MN9D cells.

Original languageEnglish
Pages (from-to)274-282
Number of pages9
JournalExperimental Neurology
Volume159
Issue number1
DOIs
Publication statusPublished - 1999 Jan 1

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Caspase 2
Cell Death
Calcium
Cell Line
Proteins
Necrosis
Emetine
Mitochondrial Swelling
bcl-2-Associated X Protein
Caspase Inhibitors
Staurosporine
Heterochromatin
Fura-2
In Situ Nick-End Labeling
Dactinomycin
DNA Fragmentation
Cycloheximide
Caspases
Mesencephalon
Fluorescent Dyes

All Science Journal Classification (ASJC) codes

  • Neurology
  • Developmental Neuroscience

Cite this

Choi, Won Seok ; Canzoniero, L. M.T. ; Sensi, S. L. ; O'Malley, Karen L. ; Gwag, Byung J. ; Sohn, Seonghyang ; Kim, Ji Eun ; Oh, Tae H. ; Lee, Eunhee B. ; Oh, Young Jun. / Characterization of MPP+-induced cell death in a dopaminergic neuronal cell line : Role of macromolecule synthesis, cytosolic calcium, caspase, and Bcl-2-related proteins. In: Experimental Neurology. 1999 ; Vol. 159, No. 1. pp. 274-282.
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Characterization of MPP+-induced cell death in a dopaminergic neuronal cell line : Role of macromolecule synthesis, cytosolic calcium, caspase, and Bcl-2-related proteins. / Choi, Won Seok; Canzoniero, L. M.T.; Sensi, S. L.; O'Malley, Karen L.; Gwag, Byung J.; Sohn, Seonghyang; Kim, Ji Eun; Oh, Tae H.; Lee, Eunhee B.; Oh, Young Jun.

In: Experimental Neurology, Vol. 159, No. 1, 01.01.1999, p. 274-282.

Research output: Contribution to journalArticle

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T1 - Characterization of MPP+-induced cell death in a dopaminergic neuronal cell line

T2 - Role of macromolecule synthesis, cytosolic calcium, caspase, and Bcl-2-related proteins

AU - Choi, Won Seok

AU - Canzoniero, L. M.T.

AU - Sensi, S. L.

AU - O'Malley, Karen L.

AU - Gwag, Byung J.

AU - Sohn, Seonghyang

AU - Kim, Ji Eun

AU - Oh, Tae H.

AU - Lee, Eunhee B.

AU - Oh, Young Jun

PY - 1999/1/1

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N2 - 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 human bcl-2 (MN9D/Bcl-2), its C-terminal deletion mutant (MN9D/Bcl-2Δ22), murine bax (MN9D/Bax), or a control vector (MN9D/Neo). As determined by electron microscopy and TUNEL assay, MN9D/Neo cells exposed to MPP+ underwent a cell death that was characterized by mitochondrial swelling and irregularly scattered heterochromatin without accompanying DNA fragmentation. However, cell swelling typically seen in necrosis did not appear. To examine the biochemical events associated with MPP+-induced cell death, various analyses were conducted. Addition of a broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (50-400 μM) or Boc-aspartyl(OMe)-fluoromethylketone (50-200 μM) did not attenuate MPP+- induced cell death while the same treatment protected MN9D/Neo cells against staurosporine-induced apoptotic cell death. Concurrent treatment with an inhibitor of macromolecule synthesis such as cycloheximide, emetine, or actinomycin D blocked MPP+-induced cell death, suggesting that new protein synthesis is required as demonstrated in many apoptotic cell death. The level of cytosolic calcium in MN9D/Neo cells was unchanged over 24 h following MPP+ treatment, as monitored by means of the fluorescent probe Fura-2. Western blot analysis indicated that expression level of proapoptotic protein, Bax was not significantly altered after MPP+ treatment. In this death paradigm, overexpression of Bcl-2 but not its C-terminal deletion mutant attenuated MPP+-induced cell death whereas overexpression of Bax had no effect. Taken together, these data indicate that (i) MPP+ induces a distinct form of cell death which resembles both apoptosis and necrosis; and (ii) full-length Bcl-2 counters MPP+-induced morphological changes and cell death via a mechanism that is dependent on de novo protein synthesis but independent of cytosolic calcium changes, Bax expression, and/or activation of caspase(s) in MN9D cells.

AB - 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 human bcl-2 (MN9D/Bcl-2), its C-terminal deletion mutant (MN9D/Bcl-2Δ22), murine bax (MN9D/Bax), or a control vector (MN9D/Neo). As determined by electron microscopy and TUNEL assay, MN9D/Neo cells exposed to MPP+ underwent a cell death that was characterized by mitochondrial swelling and irregularly scattered heterochromatin without accompanying DNA fragmentation. However, cell swelling typically seen in necrosis did not appear. To examine the biochemical events associated with MPP+-induced cell death, various analyses were conducted. Addition of a broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (50-400 μM) or Boc-aspartyl(OMe)-fluoromethylketone (50-200 μM) did not attenuate MPP+- induced cell death while the same treatment protected MN9D/Neo cells against staurosporine-induced apoptotic cell death. Concurrent treatment with an inhibitor of macromolecule synthesis such as cycloheximide, emetine, or actinomycin D blocked MPP+-induced cell death, suggesting that new protein synthesis is required as demonstrated in many apoptotic cell death. The level of cytosolic calcium in MN9D/Neo cells was unchanged over 24 h following MPP+ treatment, as monitored by means of the fluorescent probe Fura-2. Western blot analysis indicated that expression level of proapoptotic protein, Bax was not significantly altered after MPP+ treatment. In this death paradigm, overexpression of Bcl-2 but not its C-terminal deletion mutant attenuated MPP+-induced cell death whereas overexpression of Bax had no effect. Taken together, these data indicate that (i) MPP+ induces a distinct form of cell death which resembles both apoptosis and necrosis; and (ii) full-length Bcl-2 counters MPP+-induced morphological changes and cell death via a mechanism that is dependent on de novo protein synthesis but independent of cytosolic calcium changes, Bax expression, and/or activation of caspase(s) in MN9D cells.

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