Molecular Mechanisms of Hexacarbon-Induced Axon Atrophy

描述(申请人提供)：2，5-己二酮(HD)，工业溶剂甲基正丁基酮和正己烷的神经毒性二酮代谢物，在职业暴露的人类中引起中毒性神经病。虽然轴突肿胀一直被认为是形态特征，但最近的定量形态计量学研究表明，轴突萎缩是一种特殊的、普遍存在的效应，与神经功能缺陷的发生有关。这些发现表明，萎缩是二酮诱导的神经毒性的重要病理生理成分。本研究的长期目标是确定轴突萎缩的分子机制。在当前资助期(2004年至2007年)进行的研究表明，大鼠HD中毒与运动神经丝(NF)蛋白的耗尽有关。这一效应不涉及核因子磷酸化或亚单位基因表达的变化。由于HD与NFS形成吡咯加合物，我们假设加合物干扰了流动的NFS与固定的细胞骨架聚合物相互作用的能力。因此，亚基仍然附着在运输载体Kinesin上，在蛋白质合成没有代偿性变化的情况下，Kinesin促进了NF亚基的顺行丢失。萎缩的发生是由于流动的核因子池耗尽，以及随之而来的细胞骨架周转受损。这一假说将通过以下具体目标得到验证：1)质谱学将被用来表征HD在固定和流动的氮肥池中诱导的吡咯的形成。2)吡咯的形成对基于运动蛋白的核因子亚单位转运、组装和细胞骨架转运的影响将被确定。3)HD中毒大鼠和年龄匹配的对照组大鼠外周有髓轴突中细胞骨架元素(神经纤维、微管)的含量和空间关系将被量化。4)将评估HD结构类似物(例如，3，4-二甲基2，5-HD)可预测地改变蛋白质组、形态和生化参数的能力。了解轴突萎缩在溶剂性神经毒性中的作用和机制对人类职业健康和风险评估具有广泛的意义。

DESCRIPTION (provided by applicant): 2,5-Hexanedione (HD), the neurotoxic diketone metabolite of the industrial solvents methyl n-butyl ketone and n-hexane, causes a toxic neuropathy in occupationally exposed humans. Although axonal swellings have been considered the morphological hallmark, recent quantitative morphometric studies show that axon atrophy is a specific, prevalent effect that is temoporally correlated to the development of neurological defects. These findings suggest that atrophy is an essential pathophysiological component of diketone- induced neurotoxicity. The long-term objectives of this research project are to determine the molecular mechanism of axon atrophy. Studies conducted during the current funding period (yrs 04-07) indicated that HD intoxication of rats was associated with a depletion of mobile neurofilament (NF) proteins. This effect did not involve changes in NF phosphorylation or subunit gene expression. Since HD forms pyrrole adducts with NFs, we hypothesize that adduction interferes with the ability of mobile NFs to interact with the stationary cytoskeleton polymer. As a result, the subunit remains attached to the transport vector kinesin, which in the absence of compensatory changes in protein synthesis, promotes anterograde loss of NF subunits. Atrophy occurs due to depletion of the mobile NF pool and to the ensuing impairment of cytoskeletal turnover. This hypothesis will be tested by the following Specific Aims: 1) Mass spectrometry will be used to characterize HD-induced pyrrole formation in the stationary and mobile NF pools. 2) The effects of pyrrole formation on NF subunit kinesin-based transport, assembly and cytoskeletal incorportation will be determined. 3) The content and spatial relationships among cytoskeletal elements (NFs, microtubules) will be quantified in peripheral myelinated axons of HD-intoxicated rats and age-matched controls. 4) The ability of HD structural analogs (e.g., 3,4-dimethyl 2,5-HD) to predictably alter proteomic, morphological and biochemical parameters will be evaluated. Understanding the role and mechanism of axon atrophy in solvent neurotoxicity has broad-based implications for human occupational health and risk assessment.