HNE damage of adenine nucleotide translocase in ethanol-mediated neuron apoptosis

描述（由申请方提供）：该探索性建议的总体假设是，乙醇介导的线粒体通透性转换孔复合物开放和可能的细胞凋亡激活的关键机制是4-羟基壬烯醛（HNE）加合物形成对腺嘌呤核苷酸移位酶（ANT）功能的干扰。如前所述，对HNE的关键反应是抑制ANT活性、激活线粒体通透性转换孔和最终释放促凋亡蛋白。有人提出，这是一个多因素的事件，其中包括直接修改特定的氨基酸残基的ANT和受损的相互作用与亲环素D（CyP-D）。重要性：这一建议解决了一个新的和新颖的，神经介导的，基本的机制，乙醇可能会引起神经元凋亡死亡。像这样的神经元存活控制点是最终开发新临床干预措施的主要目标。具体目标1将解决这一假设，即4-羟基壬烯醛加合物形成的腺嘌呤核苷酸转位酶的修饰是乙醇对线粒体渗透性转换孔复合物并最终诱导细胞凋亡的相关影响的机制。具体目标2将解决以下假设：通过形成4-羟基壬烯醛加合物直接修饰亲环素-D，改变与腺嘌呤核苷酸移位酶的关键相互作用，这是乙醇对线粒体渗透性转换孔复合物相关影响的一种机制。实验方法：实验方法将利用质谱法确定ANT和CyP-D上修饰的特定氨基酸残基，并通过点突变操作确定这些乙醇相关修饰的功能意义。研究还将确定这些特异性ANT和CyP-D修饰对CyP-D与ANT结合、对渗透性转换调节以及对乙醇介导的神经元死亡的敏感性的影响。实验模型将是暴露于临床相关乙醇处理方案的培养胎鼠大脑皮层神经元。 公共卫生相关性：暴露于培养的胎鼠大脑皮层神经元乙醇迅速eleranized氧化应激和增加形成的4-羟基壬烯醛（HNE）在线粒体中，这是其次是线粒体泄漏的促凋亡蛋白和凋亡性死亡。这个探索性的建议解决了一个假设，即乙醇介导的渗透性转换孔复合物（PTPC）的开放机制与孔组分腺嘌呤核苷酸移位酶（ANT）和亲环素D（CyP-D）的HNE修饰有关。因此，拟议的研究将提供深入了解的基本机制，有丝分裂毒性反应乙醇以及PTPC功能的基本机制。

DESCRIPTION (provided by applicant): The overall hypothesis of this exploratory proposal is that a key mechanism underlying ethanol-mediated opening of the mitochondrial permeability transition pore complex and possibly apoptosis activation, is perturbation of adenine nucleotide translocase (ANT) function by 4- hydroxynonenal (HNE) adduct formation. Key responses to HNE are, as previously documented, inhibition of ANT activity, activation of the mitochondrial permeability transition pore, and ultimate release of pro-apoptotic proteins. It is proposed that this occurs as a multifactorial event which includes direct modification of specific amino acid residues of ANT and its impaired interactions with cyclophilin D (CyP-D). Significance: This proposal address a new and novel, mitochondrially-mediated, fundamental mechanism by which ethanol may elicit apoptotic death of neurons. Neuron survival control points such as this are prime targets for the ultimate development of new clinical interventions. Specific Aim 1 will address the hypothesis that modifications of adenine nucleotide translocase by 4- hydroxynonenal adduct formation is a mechanism underlying ethanol-related effects on the mitochondrial permeability transition pore complex and ultimately on induction of apoptosis. Specific Aim 2 will address the hypothesis that direct modifications of cyclophilin-D by 4- hydroxynonenal adduct formation alter critical interactions with adenine nucleotide translocase and that this is one mechanism underlying ethanol-related effects on the mitochondrial permeability transition pore complex. Experimental Approaches: The experimental approaches will utilize Mass Spectrometry to determine specific amino acid residues on ANT and CyP-D that are modified and point-mutational manipulations to establish the functional significance of these ethanol-related modifications. Studies will also establish effects of these specific ANT and CyP-D modifications on CyP-D binding to ANT, on permeability transition regulation, and on sensitivity to ethanol-mediated neuron death. The experimental model will be cultured fetal rat cerebral cortical neurons exposed to clinically relevant ethanol treatment regimens. PUBLIC HEALTH RELEVANCE: Exposure of cultured fetal rat cerebral cortical neurons to ethanol rapidly elicits oxidative stress and increased formation of 4-hydroxynonenal (HNE) in mitochondria, which is followed by mitochondrial leakage of pro-apoptotic proteins and apoptotic death. This exploratory proposal addresses the hypothesis that a mechanism underlying ethanol-mediated opening of the permeability transition pore complex (PTPC) is connected to HNE modifications of the pore components, adenine nucleotide translocase (ANT) and cyclophilin D (CyP-D). Thus, the proposed studies will provide insight into basic mechanisms underlying mitotoxic responses to ethanol as well as fundamental mechanisms of PTPC function.