MOLECULAR MECHANISMS OF HEXACARBON-INDUCED AXON ATROPHY

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代谢物，可引起远端轴索病变。巨大的神经丝状轴突肿胀一直被认为是神经纤维性轴索肿胀的形态特征，因此一直是机制研究的焦点。然而，已发表的证据表明，轴突萎缩，这也发生在伽马二酮中毒，可能是一个重要的致病事件。PI的实验室在当前资助期间进行的定量形态计量学研究表明，HD中毒大鼠周围神经中的纤维萎缩是一种特殊的、普遍的影响，与神经传导异常有关，并先于行为缺陷。这些发现提示萎缩是伽马二酮所致神经毒性的重要病理生理成分。平行的分子检测表明，神经丝(NF)基因表达和神经含量的减少是HD暴露大鼠轴突口径减少的直接原因。相反，我们的研究表明，巨大的轴突肿胀是与低剂量HD暴露有关的一种附带现象。本研究项目的长期目标是评估轴突萎缩的神经毒理学相关性，并确定相应的分子机制。HD是如何导致核因子合成选择性减少的尚不清楚。过去十年的研究表明，成熟轴突的口径是通过靶源性神经营养因子对NF表达的影响来维持的。因此，我们假设伽玛二酮诱导的轴突萎缩是由神经营养因子信号转导中断所致。这一假说将根据以下具体目的进行验证：(1)在伽马二酮中毒大鼠周围神经轴上检测神经营养因子(BDNF，NGF)信号复合体的形成、激活和逆行转运。(2)伽玛二酮对神经营养素受体结合和神经元内化的影响将被确定。(3)观察γ-二酮中毒对神经营养素-受体复合体刺激RAS信号转导通路、激活MAP激酶及随后的核转位的影响。这项拟议的研究代表了中毒性轴索病变机制的一个新的研究领域，并对与纤维萎缩相关的获得性或遗传性人类神经病具有广泛的影响。此外，我们的研究可能会提出基于神经营养素干预或替代的新药物疗法。

2,5-Hexanedione (HD), the neurotoxic gamma-diketone metabolite of industrial solvents methyl n-butyl ketone and n-hexane, causes distal axonopathy. Giant neurofilamentous axonal swellings have been considered the morphologic hallmark and, accordingly, have been the focus of mechanistic research. However, published evidence suggests that axon atrophy, which also occurs in response to gamma-diketone intoxication, might be a significant pathogenic event. Quantitative morphometric studies conducted in the PI's laboratory during the current funding period showed that fiber atrophy in peripheral nerve of HD-intoxicated rats was a specific, prevalent effect that was temporally correlated to nerve conduction abnormalities and preceded behavioral defects. These findings implied atrophy was an essential pathophysiologic component of gamma-diketone-induced neurotoxicity. Parallel molecular determinations suggested that a reduction in neurofilament (NF) genetic expression and nerve content were the immediate cause of decreased axon caliber in HD exposed rats. In contrast, our research indicated giant axonal swellings were an epiphenomenon related to low dose HD exposure. The long-term objectives of this research project are to evaluate the neurotoxicological relevance of axonal atrophy and determine the corresponding molecular mechanism. How HD induces a selective reduction in NF synthesis is unknown. Research over the past decade has shown that mature axon caliber is maintained by target-derived neurotrophic factor influence on NF expression. Therefore, we hypothesize gamma-diketone-induced axon atrophy is produced by disruption of neuronal trophic factor signaling. This hypothesis will be tested according to the following specific aims: (1) The formation, activation and retrograde transport of neurotrophin (BDNF, NGF) signal complexes will be measured in peripheral nerve axis of gamma-diketone intoxicated rats. (2) gamma-Diketone influences on neurotrophin-trk receptor binding and neuronal internalization will be determined. (3) Assess the effects of gamma-diketone intoxication on neurotrophin-receptor complex stimulation of the Ras signal transduction pathway and activation of MAP kinase and subsequent nuclear translocation. The proposed research represents a new area of investigation into mechanisms of toxic axonopathies and has broad-based implications for acquired or inherited human neuropathies associated with fiber atrophy. In addition, our studies might suggest novel pharmacotherapies based on neurotrophin intervention or replacement.