Molecular Mechanisms of Hexacarbon-Induced Axon Atrophy

描述（由申请人提供）：2,5-己二酮（HD）是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变。虽然轴突肿胀被认为是形态学的标志，但最近的定量形态学研究表明，轴突萎缩是一种特定的、普遍的效应，与神经系统缺陷的发展暂时相关。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组成部分。本研究项目的长期目标是确定轴突萎缩的分子机制。在当前资助期（04-07年）进行的研究表明，HD中毒大鼠与移动神经丝（NF）蛋白的消耗有关。这种影响不涉及NF磷酸化或亚基基因表达的变化。由于HD与NFs形成吡咯加合物，我们假设内合物干扰了移动NFs与固定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激酶上，这在蛋白质合成缺乏代偿性变化的情况下，促进了NF亚基的顺行损失。萎缩的发生是由于可移动的NF池耗竭和随后的细胞骨架周转受损。这一假设将通过以下具体目标进行验证：1)质谱分析将用于表征固定和移动NF池中hd诱导的吡咯形成。2)将确定吡咯形成对NF亚基运动蛋白转运、组装和细胞骨架整合的影响。3)定量分析hd中毒大鼠和年龄匹配对照组外周髓鞘轴突细胞骨架元件（nf、微管）的含量和空间关系。4) 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学，形态学和生化参数的能力将被评估。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.