Blockade of PKCδ proteolytic activation by loss of function mutants rescues mesencephalic dopaminergic neurons from methylcyclopentadienyl manganese tricarbonyl (MMT)-induced apoptotic cell death
Blockade of PKCδ proteolytic activation by loss of function mutants rescues mesencephalic dopaminergic neurons from methylcyclopentadienyl manganese tricarbonyl (MMT)-induced apoptotic cell death
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DOI：
10.1196/annals.1332.017
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发表时间：
2004-01-01
期刊：
影响因子：
--
通讯作者：
Kanthasamy, AG
中科院分区：
文献类型：
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作者：
Anantharam, V;Kitazawa, M;Kanthasamy, AG
甲基三羰基锰（MMT）作为汽油添加剂的使用引起了人们对健康的关注，并增加了人们对了解锰的神经毒性作用的兴趣。长期接触无机锰会导致锰中毒，这是一种有点类似于帕金森病的神经系统疾病。然而，有机锰化合物MMT诱导多巴胺能神经元细胞神经毒性的细胞机制尚不清楚。因此，我们系统地研究了暴露于3-200 μ M MMT中脑多巴胺能神经元（N27）细胞凋亡细胞信号转导事件。MMT处理导致N27细胞中活性氧产生和细胞死亡的时间和剂量依赖性增加。在细胞死亡之前，依次激活的细胞凋亡依赖的促凋亡事件，包括细胞色素c释放，半胱天冬酶-3激活，和DNA片段化，表明细胞凋亡依赖的凋亡级联反应主要触发MMT诱导的凋亡性细胞死亡。重要的是，MMT诱导蛋白激酶C δ（PKC δ）的蛋白水解裂解，导致激酶活性持续增加。用100 μ M Z-VAD-FMK和100 μ M Z-DEVD-FMK处理可抑制PKC δ的蛋白水解激活，表明caspase-3介导PKC δ的蛋白水解激活。用100 μ M Z-DEVD-FMK和5 μ M rottlerin（一种PKC δ抑制剂）预处理也显著减弱MMT诱导的DNA断裂。此外，激酶失活显性负性PKC δ（K376 R）突变体或半胱天冬酶切割抗性PKC δ（D327 A）突变体的过表达拯救了N27细胞免于MMT诱导的DNA片段化。总的来说，这些结果表明，在MMT诱导的多巴胺能变性中，神经细胞依赖性凋亡级联通过蛋白水解激活PKC δ介导凋亡，并表明PKC δ可作为帕金森相关神经系统疾病的有吸引力的治疗靶点。
The use of methytcyclopentadienyl manganese tricarbonyl (MMT) as a gasoline additive has raised health concerns and increased interest in understanding the neurotoxic effects of manganese. Chronic exposure to inorganic manganese causes Manganism, a neurological disorder somewhat similar to Parkinson's disease. However, the cellular mechanism by which MMT, an organic manganese compound, induces neurotoxicity in dopaminergic neuronal cells remains unclear. Therefore, we systematically investigated apoptotic cell-signaling events following exposure to 3-200 mu M MMT in mesencephalic dopaminergic neuronal (N27) cells. MMT treatment resulted in a time- and dose-dependent increase in reactive oxygen species generation and cell death in N27 cells. The cell death was preceded by sequential activation of mitochondrial-dependent proapoptotic events including cytochrome c release, caspase-3 activation, and DNA fragmentation, indicating that the mitochondrial-dependent apoptotic cascade primarily triggers MMT-induced apoptotic cell death. Importantly, MMT induced proteolytic cleavage of protein kinase C delta (PKC delta), resulting in persistently increased kinase activity. The proteolytic activation of PKC delta was suppressed by treatment with 100 mu M Z-VAD-FMK and 100 mu M Z-DEVD-FMK, suggesting that caspase-3 mediates the proteolytic activation of PKC delta. Pretreatment with 100 mu M Z-DEVD-FMK and 5 mu M rottlerin (a PKC delta inhibitor) also significantly attenuated MMT-induced DNA fragmentation. Furthermore, overexpression of either the kinase inactive dominant negative PKC delta(K376R) mutant or the caspase cleavage resistant PKC delta(D327A) mutant rescued N27 cells from MMT-induced DNA fragmentation. Collectively, these results demonstrate that the mitochondrial-dependent apoptotic cascade mediates apoptosis via proteolytic activation of PKC delta in MMT-induced dopaminergic degeneration and suggest that PKC delta may serve as an attractive therapeutic target in Parkinson-related neurological diseases.