ENVIRONMENTAL NEUROTOXICANTS & THE AXONAL CYTOSKELETON

这个项目的广泛的长期目标是阐明 环境和职业的分子作用机制 在实验动物中诱导轴突变性的神经毒素， 伙计 本提案审查了这一组的一个重要子类， 神经丝（NF）神经毒素，包括正己烷、2，5-己二酮 （2，5-HD）、二硫化碳（CS2）、β，β-亚氨基二丙腈（IDPN）、 丙烯酰胺。 这些代理商产生细胞骨架解体和NF 在敏感神经纤维内积聚。 NF神经毒素代表了 一个重大的公共卫生危害，并鉴于其巨大的化学和 结构多样性，重要的是要确定共同的步骤， 各自的机制。 这些信息将是非常有价值的， 预测未经测试或新合成的化学物质也会产生类似的效果。 具体目标包括：i）结合的表征和定量 2，5-HD、CS2、IDPN（或其代谢物）和丙烯酰胺对轴突的 细胞骨架蛋白; ii）确定这种衍生化是否 局限于NF蛋白内的特定位点或随机地 iii）阐明在体外和体内的作用 神经毒素暴露对NF-NF和NF-微管（MT）相互作用的影响以及对 轴突细胞骨架结构;和iv）确定分化的细胞骨架结构。 远端与近端轴突对共价结合的吸收的敏感性 通过NF神经毒素衍生化。 体外轴突细胞骨架和整体 将采用动物模型系统。 结合研究将利用高 比活度[14 C]标记的NF神经毒素。 分析技术 将包括细胞骨架蛋白的凝胶电泳，荧光照相术， 和凝胶切片的闪烁计数。 蛋白质结合位点将是 通过化学和酶促裂解，然后通过HPLC肽进行评估 作图和自动肽测序。 蛋白质加合物将是 通过质谱法表征。 NF-NF相互作用将通过以下方式进行评估： 测量NF蛋白重组和NF网络形成的动力学 使用来自治疗大鼠的天然NF。 NF-MT亲和力将用以下方法研究： 利用分离的NF和[32 P]标记的MT的印迹测定。 将在器官型神经培养中检查细胞骨架重组 使用视频增强和微分干涉对比光 免疫荧光显微镜和高压电子显微镜 显微镜 放射性标记NF的区域摄取和共价结合 神经毒素将沿着治疗大鼠的视神经进行沿着评估， 器官型神经培养 调查结果将提供实质性进展 为这类重要的 神经毒素

The broad, long-term objective of this project is to elucidate the molecular mechanisms of action of environmental and occupational neurotoxins which induce axonal degeneration in experimental animals and man. This proposal examines an important subclass of this group, the neurofilament (NF) neurotoxins, which includes n-hexane, 2,5-hexanedione (2,5-HD), carbon disulfide (CS2), beta, beta-iminodipropionitrile (IDPN), an acrylamide. These agents produce cytoskeletal disorganization and NF accumulation within susceptible nerve fibers. The NF neurotoxins represent a significant public health hazard, and in view of their great chemical and structural diversity, it is important to identify common steps in their respective mechanisms. Such information would be of great value in predicting similar effects from untested or newly synthesized chemicals. Specific aims include: i) Characterization and quantitation of the binding of 2,5-HD, CS2, IDPN (or its metabolites), and acrylamide to axonal cytoskeletal proteins; ii) Determination of whether such derivatization is limited to specific sites within the NF proteins or is randomly distributed; iii) Elucidation of the effects of in vitro and in vivo neurotoxin exposure on NF-NF and NF-microtubule (MT) interactions and on axonal cytoskeletal structures; and iv) Determination of the differential susceptibility of the distal vs. proximal axon to uptake of and covalent derivatization by NF neurotoxins. In vitro axonal cytoskeletal and whole animal model systems will be employed. Binding studies will utilize high specific activity [14C]-labelled NF neurotoxins. Analytical techniques will include gel electrophoresis of cytoskeletal proteins, fluorography, and scintillation counting of gel slices. Sites of protein binding will be assessed by chemical and enzymatic cleavage followed by HPLC peptide mapping and automated peptide sequencing. Protein adducts will be characterized by mass spectrometry. NF-NF interactions will be assessed by measuring the kinetics of NF protein reassembly and NF network formation using native NFs from treated rats. NF-MT affinity will be studied with blotting assays utilizing isolated NFs and [32P]-labelled MTs. Cytoskeletal reorganization will be examined in organotypic nerve cultures using video-enhanced and differential interference contrast light microscopy, immunofluorescence microscopy, and high voltage electron microscopy. Regional uptake and covalent binding of radiolabelled NF neurotoxins will be assessed along the optic nerve of treated rats and in organotypic nerve cultures. Findings will provide substantial progress towards defining mechanisms of action for this important class of neurotoxins.