Molecular Mechanisms of Hexacarbon-Induced Axon Atrophy

描述（由申请方提供）：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，可导致职业暴露人群出现毒性神经病。虽然轴突萎缩一直被认为是形态学标志，但最近的定量形态学研究表明，轴突萎缩是一种特异性的、普遍的效应，与神经系统缺陷的发展在时间上相关。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学组成部分。本研究项目的长期目标是确定轴突萎缩的分子机制。在当前资助期间（2004 - 2007年）进行的研究表明，大鼠HD中毒与移动的神经丝（NF）蛋白耗竭相关。这种效应不涉及NF磷酸化或亚基基因表达的变化。由于HD与NF形成吡咯加合物，我们假设加合干扰了移动的NF与固定的细胞骨架聚合物相互作用的能力。因此，该亚基仍然附着在转运载体驱动蛋白上，在蛋白质合成缺乏代偿性变化的情况下，促进NF亚基的顺行丢失。由于移动的NF池的耗尽和随后的细胞骨架周转的损害而发生萎缩。将通过以下特定目的检验该假设：1）将使用质谱法表征固定和移动的NF合并液中HD诱导的吡咯形成。2)将确定吡咯形成对基于NF亚基驱动蛋白的转运、组装和细胞骨架整合的影响。3)将在HD中毒大鼠和年龄匹配的对照组的外周有髓轴突中定量细胞骨架元素（NF，微管）之间的含量和空间关系。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。了解轴突萎缩在溶剂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.