烃类溶剂代谢产物γ-双酮与stathmin相互作用在烃类溶剂致轴索病变的机制

Repeated occupational exposure to aliphatic hydrocarbon solvents (e.g., n-hexane; methyl n-butyl ketone) and aromatic hydrocarbon solvents (e.g.,1,2-diethylbenzene; 1,2,4-triethylbenzene) induce axonal degeneration (axonopathy) in both the peripheral nervous system (PNS) and the central nervous system (CNS). 2,5-Hexanedione (2,5-HD) and 1,2-diacetylbenzene (1,2-DAB), the active metabolites of n-hexane and 1,2-diethylbenzene (1,2-DAB), respectively, are γ-diketones that rapidly react with lysyl residues in proteins to form orange-colored pyrrole adducts (for 2,5-HD) and blue colored indole adducts (for 1,2-DAB). Although it is widely believed that the accumulation of polymerized adducts induce axonopathy, the precise molecular mechanisms have yet to be determined. Our preliminary data show that 1,2-DAB and 2,5-HD respectively induce neurofilament-filled axonal swellings in proximal and distal regions of nerve fibers while their protein-nonreactive isomers, 1,3-DAB and 2,4-HD, have no effect on nerve fiber integrity. Furthermore, we found significant higher levels of the protein stathmin in the brain tissue of 1,2-DAB-intoxicated mice when we compared the proteomic data from 1,2-DAB treated mice with 1,3-DAB or saline-treated animals. Since stathmin has the highest lysine content of all axonal proteins, and destabilizes microtubules required for transport of axonal proteins, we hypothesized that stathmin is the primary molecular target for γ-diketones and their inactivation responsible for the induction axonopathy. We seek grant support to confirm that stathmin is a key target of γ-diketones and to explore how stathmin binding initiates axonal deficits. Our strategy will utilize advanced analytical techniques (dynamics of molecular interactions, phosphoproteomics, immunocytochemistry) and focus on the very early (pre-axonopathy) molecular events and responses, thereby avoid the cascade of complex events of subsequent molecular events that eventuate in axonal breakdown. Since axonal degeneration is a common response of the human nervous system to adverse factors (occupational, drug, metabolic, genetic), we anticipate the results of our study will have wide relevance to human neurological and neurodegenerative disease.

正己烷和1,2-二乙基苯的慢性暴露诱发中枢和周围神经轴索病变。二者的活性代谢物2,5-HD和1,2-DAB均为γ-双酮，与赖氨酸的ε氨基发生加合反应。尽管广泛认为γ-双酮加合反应的累积最终诱发轴索病变，然而这一过程如何发生仍然缺乏依据。申请人前期研究发现1,2-DAB暴露的小鼠相对其异构体1,3-DAB和空白诱发轴索病变，且1,2-DAB暴露显著上调微管解聚蛋白stathmin的表达。鉴于stathmin在轴索蛋白中赖氨酸含量最高，也是关键的微管调节蛋白，我们推测其为γ-双酮致轴索病变的靶标蛋白。申请人拟研究γ-双酮与stathmin的相互作用，预测二者的结合位点，验证和检测γ-双酮对stathmin的表达及翻译后修饰的作用，探讨γ-双酮对stathmin的微管调节功能的影响。本研究从γ-双酮的化学结构出发，研究γ-双酮中毒超早期的分子事件，为烃类溶剂致轴索神经病变的防治提供科学依据。

脂肪烃类溶剂（如正己烷、甲基正丁基酮）和芳香烃（如1, 2-二乙基苯， 1, 2, 4-三乙基苯）溶剂慢性中毒引发周围神经（PNS）和中枢神经（CNS）损害，病理学发现其轴索发生包括肿胀、脱鞘、回缩等一系列退行性病变。目前治疗慢性烃类溶剂尚无特效解毒药，且中枢神经损害不可逆转。本研究从脂肪烃和芳香烃毒性代谢产物γ-双酮与蛋白加合反应入手，先研究γ-双酮与关键细胞骨架蛋白的作用位点，再研究γ-双酮暴露的影响的蛋白表达谱和磷酸化修饰谱，最后在人群中验证γ-双酮加合蛋白水平、关键细胞骨架蛋白水平、与周围神经病变的相关性。本研究取得了丰硕的成果，发表了SCI期刊文章3篇，培养硕士生2名，培养博士后1名。本研究所得结果在已经应用于临床研究，即探索γ-双酮加合蛋白与糖尿病远端对称多发性神经病变以及糖尿病心脏自主神经病变之间的关系中。