MITOCHONDRIAL PERMEABILITY TRANSITION IN NEUROTOXICITY

本提案中概述的研究目的是确定线粒体通透性转换（MPT）在神经细胞对二硝基苯暴露影响的选择性脆弱性中的作用。 二硝基苯（DNB）广泛用作染料、塑料和炸药制造中的工业中间体，并且在具有高能量需求的核中诱导脑干神经胶质血管病变。线粒体被认为是DNB诱导的氧化应激的主要靶点，并且线粒体功能障碍程度的差异可以部分解释CNS细胞对DNB暴露的神经毒性作用的选择性脆弱性。 本研究的目的是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）评价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换（MPT）的能力;（2）确定细胞氧化还原电位的变化程度和还原的吡啶核苷酸的亚细胞分布（3）确定Bcl-2和Bcl-XL的组成型或诱导型表达是否影响原代神经元和星形胶质细胞对MPT的敏感性，并建立Bcl-2和Bcl-XL在分离的线粒体中保护免受DNB诱导的MPT的相对效力。 为了检验这些假设，将通过激光扫描共聚焦显微镜评价原代培养物中的神经元和星形胶质细胞，以获得暴露于DNB后MPT的证据。 设计用于确定任何观察到的对MPT的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和抗凋亡Bcl-2家族蛋白保护免受DNB诱导的MPT的能力。

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.