Caspase-dependent and -independent cell death pathways in primary cultures of mesencephalic dopaminergic neurons after neurotoxin treatment

Baek S. Han, Hyun Seung Hong, Won Seok Choi, George J. Markelonis, Tae H. Oh, Young Jun Oh

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Abstract

Although the cause of neuronal death in Parkinson's disease (PD) is mainly unknown, growing evidence suggests that both apoptotic and non-apoptotic death may occur in PD. Using primary cultures of mesencephalic dopaminergic neurons and the MN9D dopaminergic neuronal cell line, we attempted to evaluate specifically the existence of the mitochondrial apoptotic pathway, focusing on the mitochondrial release of cytochrome c to the activation of the caspases after 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenylpyridinium (MPP+) treatment. Both immunofluorescent labeling and immunoblot analysis indicated mitochondrial release of cytochrome c into the cytosol after 6-OHDA or MPP+ treatment. However, the appearance of activated caspase-3 immunoreactivity in tyrosine hydroxylase (TH)-positive neurons was detected only after 6-OHDA. Immunoblot and biochemical analysis also confirmed that activation of both caspase-9 and caspase-3 was induced by 6-OHDA, but not by MPP+. Consequently, cotreatment with a caspase inhibitor (zVAD-fmk) or with an antioxidant (N-acetylcysteine) not only deterred 6-OHDA-induced loss of TH-positive neurons but also abolished the appearance of activated caspase-3 in TH-positive neurons. In contrast, the same treatment did not spare MPP+-treated TH-positive neurons. Interestingly, a reconstitution assay indicated that the addition of ATP to the cytosolic fraction obtained from MPP+-treated cells was sufficient to activate both caspase-9 and caspase-3. Taken together, our results indicate that distinct mechanisms underlie neurotoxin-induced cell death. They also suggest that, after mitochondrial release of cytochrome c in dopaminergic neurons after neurotoxin treatment, intracellular levels of ATP may constitute a critical factor in determining whether a neuron will die by a caspase-dependent or -independent pathway.

Original languageEnglish
Pages (from-to)5069-5078
Number of pages10
JournalJournal of Neuroscience
Volume23
Issue number12
Publication statusPublished - 2003 Jun 15

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Oxidopamine
Dopaminergic Neurons
Neurotoxins
Caspases
Cell Death
Tyrosine 3-Monooxygenase
Caspase 3
Neurons
Cytochromes c
Caspase 9
Parkinson Disease
Adenosine Triphosphate
Caspase 6
1-Methyl-4-phenylpyridinium
Caspase Inhibitors
Acetylcysteine
Cytosol
Cause of Death
Antioxidants
Cell Line

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)

Cite this

Han, Baek S. ; Hong, Hyun Seung ; Choi, Won Seok ; Markelonis, George J. ; Oh, Tae H. ; Oh, Young Jun. / Caspase-dependent and -independent cell death pathways in primary cultures of mesencephalic dopaminergic neurons after neurotoxin treatment. In: Journal of Neuroscience. 2003 ; Vol. 23, No. 12. pp. 5069-5078.
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abstract = "Although the cause of neuronal death in Parkinson's disease (PD) is mainly unknown, growing evidence suggests that both apoptotic and non-apoptotic death may occur in PD. Using primary cultures of mesencephalic dopaminergic neurons and the MN9D dopaminergic neuronal cell line, we attempted to evaluate specifically the existence of the mitochondrial apoptotic pathway, focusing on the mitochondrial release of cytochrome c to the activation of the caspases after 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenylpyridinium (MPP+) treatment. Both immunofluorescent labeling and immunoblot analysis indicated mitochondrial release of cytochrome c into the cytosol after 6-OHDA or MPP+ treatment. However, the appearance of activated caspase-3 immunoreactivity in tyrosine hydroxylase (TH)-positive neurons was detected only after 6-OHDA. Immunoblot and biochemical analysis also confirmed that activation of both caspase-9 and caspase-3 was induced by 6-OHDA, but not by MPP+. Consequently, cotreatment with a caspase inhibitor (zVAD-fmk) or with an antioxidant (N-acetylcysteine) not only deterred 6-OHDA-induced loss of TH-positive neurons but also abolished the appearance of activated caspase-3 in TH-positive neurons. In contrast, the same treatment did not spare MPP+-treated TH-positive neurons. Interestingly, a reconstitution assay indicated that the addition of ATP to the cytosolic fraction obtained from MPP+-treated cells was sufficient to activate both caspase-9 and caspase-3. Taken together, our results indicate that distinct mechanisms underlie neurotoxin-induced cell death. They also suggest that, after mitochondrial release of cytochrome c in dopaminergic neurons after neurotoxin treatment, intracellular levels of ATP may constitute a critical factor in determining whether a neuron will die by a caspase-dependent or -independent pathway.",
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Caspase-dependent and -independent cell death pathways in primary cultures of mesencephalic dopaminergic neurons after neurotoxin treatment. / Han, Baek S.; Hong, Hyun Seung; Choi, Won Seok; Markelonis, George J.; Oh, Tae H.; Oh, Young Jun.

In: Journal of Neuroscience, Vol. 23, No. 12, 15.06.2003, p. 5069-5078.

Research output: Contribution to journalArticle

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T1 - Caspase-dependent and -independent cell death pathways in primary cultures of mesencephalic dopaminergic neurons after neurotoxin treatment

AU - Han, Baek S.

AU - Hong, Hyun Seung

AU - Choi, Won Seok

AU - Markelonis, George J.

AU - Oh, Tae H.

AU - Oh, Young Jun

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N2 - Although the cause of neuronal death in Parkinson's disease (PD) is mainly unknown, growing evidence suggests that both apoptotic and non-apoptotic death may occur in PD. Using primary cultures of mesencephalic dopaminergic neurons and the MN9D dopaminergic neuronal cell line, we attempted to evaluate specifically the existence of the mitochondrial apoptotic pathway, focusing on the mitochondrial release of cytochrome c to the activation of the caspases after 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenylpyridinium (MPP+) treatment. Both immunofluorescent labeling and immunoblot analysis indicated mitochondrial release of cytochrome c into the cytosol after 6-OHDA or MPP+ treatment. However, the appearance of activated caspase-3 immunoreactivity in tyrosine hydroxylase (TH)-positive neurons was detected only after 6-OHDA. Immunoblot and biochemical analysis also confirmed that activation of both caspase-9 and caspase-3 was induced by 6-OHDA, but not by MPP+. Consequently, cotreatment with a caspase inhibitor (zVAD-fmk) or with an antioxidant (N-acetylcysteine) not only deterred 6-OHDA-induced loss of TH-positive neurons but also abolished the appearance of activated caspase-3 in TH-positive neurons. In contrast, the same treatment did not spare MPP+-treated TH-positive neurons. Interestingly, a reconstitution assay indicated that the addition of ATP to the cytosolic fraction obtained from MPP+-treated cells was sufficient to activate both caspase-9 and caspase-3. Taken together, our results indicate that distinct mechanisms underlie neurotoxin-induced cell death. They also suggest that, after mitochondrial release of cytochrome c in dopaminergic neurons after neurotoxin treatment, intracellular levels of ATP may constitute a critical factor in determining whether a neuron will die by a caspase-dependent or -independent pathway.

AB - Although the cause of neuronal death in Parkinson's disease (PD) is mainly unknown, growing evidence suggests that both apoptotic and non-apoptotic death may occur in PD. Using primary cultures of mesencephalic dopaminergic neurons and the MN9D dopaminergic neuronal cell line, we attempted to evaluate specifically the existence of the mitochondrial apoptotic pathway, focusing on the mitochondrial release of cytochrome c to the activation of the caspases after 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenylpyridinium (MPP+) treatment. Both immunofluorescent labeling and immunoblot analysis indicated mitochondrial release of cytochrome c into the cytosol after 6-OHDA or MPP+ treatment. However, the appearance of activated caspase-3 immunoreactivity in tyrosine hydroxylase (TH)-positive neurons was detected only after 6-OHDA. Immunoblot and biochemical analysis also confirmed that activation of both caspase-9 and caspase-3 was induced by 6-OHDA, but not by MPP+. Consequently, cotreatment with a caspase inhibitor (zVAD-fmk) or with an antioxidant (N-acetylcysteine) not only deterred 6-OHDA-induced loss of TH-positive neurons but also abolished the appearance of activated caspase-3 in TH-positive neurons. In contrast, the same treatment did not spare MPP+-treated TH-positive neurons. Interestingly, a reconstitution assay indicated that the addition of ATP to the cytosolic fraction obtained from MPP+-treated cells was sufficient to activate both caspase-9 and caspase-3. Taken together, our results indicate that distinct mechanisms underlie neurotoxin-induced cell death. They also suggest that, after mitochondrial release of cytochrome c in dopaminergic neurons after neurotoxin treatment, intracellular levels of ATP may constitute a critical factor in determining whether a neuron will die by a caspase-dependent or -independent pathway.

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