Elucidation of multiple actions of reactive nitrogen species in degeneration of midbrain dopaminergic neurons

这项研究特别参考了活性氮物种和相关化合物的作用，探讨了氧化应激在诱导中脑黑质多巴胺能神经元选择性变性中的作用，这是帕金森病的病理特征。(1)脂多糖激活培养的中脑片微痛可引起多巴胺能神经元变性，其机制可能与诱导型一氧化氮合酶(NO)表达增加和NO的产生有关。超氧化物歧化不能抑制神经元死亡的诱导，而JNK抑制剂和α-生育酚保护多巴胺神经元而不影响NO的产生。另一方面，在中脑片培养中应用凝血酶引起的多巴胺能神经元变性与多个MAPK家族成员的激活以及随后NO合成酶的表达增加有关。这些酶的抑制以及小胶质细胞的枯竭显著抑制了多巴胺能神经退行性变。因此，凝血酶可能通过激活小胶质细胞产生选择性多巴胺能神经毒性，增加NO的产生。(2)酪氨酸与NO相关分子反应生成的3-硝基酪氨酸(3-NT)可诱导培养的中脑多巴胺能神经元变性。细胞通过氨基酸转运体摄取3-NT是诱导细胞死亡的关键，3-NT促进了细胞内超氧化物的产生，提示3-NT作为细胞内超氧化物生成物促进了多巴胺能神经元的变性。(3)家族性帕金森病致病基因DJ-1的敲除使SH-SY5Y细胞易受6-OHDA、H_2O_2和NO供体等因素的影响。此外，H_2O_2处理SH-SY5Y细胞和C6细胞后，DJ-1的表达增加，提示DJ-1是氧化应激内源性保护机制的一部分。

With special reference to the actions of reactive nitrogen species and related compounds, this study addressed the roles of oxidative stress in induction of selective degeneration of midbrain nigral dopaminergic neurons, a hallmark of Parkinson disease pathology. (1) Activation of microgia in midbrain slice cultures by lipopolysaccharide induced degeneration of dopaminergic neurons, which was mediated by increases in expression of inducible nitric oxide (NO) synthase and production of NO. Dismutation of superoxide did not inhibit induction of neuronal death, whereas a JNK inhibitor and α-tocopherol protected dopaminerguc neurons without affecting NO production. On the other hand, degeneration of dopaminergic neurons induced by application of thrombin to midbrain slice cultures was associated with activation of multiple MAP kinase family members and a subsequent increase in expression of NO synthase. Inhibition of these enzymes as well as depletion of microglia markedly suppressed dopaminergic neurodegeneration. Thus, thrombin was suggested to exert selective dopaminergic neurotoxicity via microglial activation and increased NO production. (2) 3-Nitrotyrosine (3-NT), which is formed by reaction of tyrosine with NO-related molecular species, induced degeneration of midbrain dopaminergic neurons in culture. Cellular uptake of 3-NT via amino acid transporters was essential for induction of cell death, and 3-NT promoted superoxide production in cells, suggesting that 3-NT promotes degeneration of dopaminergic neurons by acting as an intracellular superoxide generator. (3) Knockdown of DJ-1, a protein encoded by a gene responsible for familial Parkinson disease, rendered SH-SY5Y cells vulnerable to several agents including 6-OHDA, H_2O_2 and an NO donor. In addition, treatment of SH-SY5Y cells and C6 cells with H_2O_2 increased expression of DJ-1, suggesting that DJ-1 functions as a part of endogenous protective mechanisms against oxidative stress.