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

描述(申请人提供)：这项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体开放和可能的细胞凋亡激活的一个关键机制是4-羟基壬醛(HNE)加合物形成对腺嘌呤核苷酸转位酶(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，并最终释放促凋亡蛋白。有人认为这是一个多因素事件，包括ANT特定氨基酸残基的直接修饰及其与亲环素D(CYP-D)的相互作用受损。意义：这项建议解决了一种新的、新颖的、线粒体介导的基本机制，通过这种机制，乙醇可能会导致神经元的凋亡性死亡。像这样的神经元存活控制点是新的临床干预措施最终发展的主要目标。具体目标1将解决这一假说，即4-羟基壬醛加合物的形成对腺嘌呤核苷酸转位酶的修饰是酒精对线粒体通透性转换孔复合体的影响，并最终诱导细胞凋亡的机制。具体目标2将解决这一假设，即亲环素-D被4-羟基壬醛加合物形成的直接修饰改变了与腺嘌呤核苷酸转位酶的关键相互作用，这是乙醇对线粒体通透性转换孔复合体产生影响的机制之一。实验方法：实验方法将利用质谱学来确定被修饰的ANT和CYP-D上的特定氨基酸残基和点突变操作，以确定这些与乙醇相关的修饰的功能意义。研究还将确定这些特定的ANT和CYP-D修饰对CYP-D与ANT的结合、通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。实验模型将培养的胎鼠大脑皮层神经元暴露于临床相关的乙醇治疗方案中。 与公共卫生相关：培养的胎鼠大脑皮层神经元暴露在乙醇中会迅速引起氧化应激，并增加线粒体中4-羟基壬烯醛(HNE)的形成，随后导致线粒体漏出促凋亡蛋白和细胞凋亡性死亡。这一探索性的建议解决了这样的假设，即乙醇介导的通透性转换孔复合体(PTPC)开放的机制与孔成分腺核苷酸转位酶(ANT)和亲环素D(CYP-D)的HNE修饰有关。因此，拟议的研究将提供对乙醇潜在的丝裂毒性反应的基本机制以及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.