MITOCHONDRIAL PERMEABILITY TRANSITION IN NEUROTOXICITY

这项建议中概述的研究的目的是确定线粒体通透性转变(MPT)在神经细胞对二硝基苯暴露的影响的选择性脆弱性中的作用。二硝基苯(DNB)被广泛用作染料、塑料和炸药制造的工业中间体，并在高能量需求的核内引起脑干胶质血管损伤。线粒体被认为是DNB诱导的氧化应激的主要靶点，线粒体功能障碍程度的差异可能部分解释了中枢神经系统细胞对DNB暴露的神经毒性效应的选择性脆弱性。为了确定神经元和星形胶质细胞对DNB暴露引起的线粒体功能障碍的不同敏感性的基础：(1)评估DNB暴露诱导培养的神经元和星形胶质细胞线粒体通透性转变(MPT)的能力；(2)确定细胞氧化还原电位的变化以及还原型吡啶核苷酸(NADH/NADPH)和谷胱甘肽(GSH)的亚细胞分布与DNB暴露的原代神经元和星形胶质细胞对MPT敏感性的相关性；(3)确定原代培养的神经元和星形胶质细胞中，Bcl2和Bclxl的组成性或诱导性表达是否影响对MPT的敏感性，并确定Bcl2和Bclxl对DNB诱导的线粒体MPT的保护作用的相对效力。为了验证这些假设，原代培养的神经元和星形胶质细胞将通过激光扫描共聚焦显微镜进行评估，以寻找暴露于DNB后发生MPT的证据。旨在确定观察到的MPT敏感性差异的基础的研究将评估DNB暴露对细胞氧化还原电位的影响，以及抗凋亡的Bcl-2家族蛋白对DNB诱导的MPT的保护能力。

The objective of the research outlined in this proposal is to establish the role of the mitochondrial permeability transition (MPT) in the selective vulnerability of neural cells to the effects of dinitrobenzene exposure. Dinitrobenzene (DNB) is widely used as an industrial intermediate in the manufacture of dyes, plastics, and explosives, and induces brain stem glio- vascular lesions in nuclei with high energy requirements. Mitochondria are thought to be a primary target for DNB-induced oxidative stress and differences in the extent of mitochondrial dysfunction may partially explain the selective vulnerability of CNS cells to the neurotoxic effects of DNB exposure. The following aims are proposed to determine the basis for the differential sensitivity of neurons and astrocytes to mitochondrial dysfunction resulting from DNB exposure: (1) Evaluate the capacity of DNB exposure to induce mitochondrial permeability transition (MPT) in cultured neurons and astrocytes; (2) Determine the extent to which changes in cellular redox potential and the subcellular distribution of reduced pyridine nucleotides (NADH/NADPH) and glutathione (GSH) correlate with sensitivity to MPT in primary neurons and astrocytes exposed to DNB; and (3) Determine whether constitutive or inducible expression of of Bcl-2 and Bcl-XL influences sensitivity to MPT in primary neurons and astrocytes and establish the relative potency of Bcl-2 and Bcl-XL to protect against DNB-induced MPT in isolated mitochondria. To test these hypotheses, neurons and astrocytes in primary culture will be evaluated by laser scanning confocal microscopy for evidence of MPT following exposure to DNB. Studies designed to determine the basis for any observed differences in sensitivity to MPT will evaluate the effects of DNB exposure on cellular redox potential and the ability of anti- apoptotic Bcl-2-family proteins to protect against DNB-induced MPT.