Role of Tyrosine in MDMA Toxicity

描述（由申请人提供）：广泛滥用的安非他明类似物，3 - 4-亚甲二氧基甲基安非他明（MDMA，Ectaline）选择性损害大脑中5-HT神经元的轴突末梢。几条证据表明，多巴胺（DA）有助于这种毒性。虽然DA清楚地产生氧化副产物，如活性氧和醌类，但仍不清楚DA衍生的氧化物质如何对5 HT末端产生选择性损伤，这是MDMA诱导的神经毒性的标志。更具体地说，DA如何在5-HT终末内积累以产生其选择性损伤的机制尚不清楚。当前提案的主要假设是，DA的氨基酸前体L-酪氨酸有助于神经退行性过程。这一假设是基于我们最近的初步数据，表明5倍增加酪氨酸细胞外浓度在体内测量后，MDMA。与DA不同，酪氨酸从外周积极转运到大脑和神经元中。虽然酪氨酸是DA神经元内DA合成的天然前体，但5 HT神经元中高浓度的酪氨酸可能具有有害后果。该建议的假设框架是，MDMA和5-HT神经元中的高温产生的氧化环境导致酪氨酸非酶促氧化为DA前体多巴。5-HT末端内的芳香族氨基酸脱羧酶（AADC）然后将DOPA脱羧为DA，导致DA的积累，并因此导致DA衍生的活性氧簇和5-HT末端内的氧化损伤。酪氨酸作为MDMA诱导毒性的介体的这种假设作用是一种新的机制，其有效地将当前现有的假设以及在某种程度上不一致的观察结果和明显的警告合成为一个有凝聚力的、理论的和可测试的框架。 具体目的是（1）证明酪氨酸在5 HT神经元内非酶促氧化为DOPA和DA，（2）表征L-酪氨酸和酪胺对MDMA和高血压诱导的氧化应激的贡献，以及（3）评估后续神经元损伤。使用培养的RN 46 A 5 HT细胞是一种新的方法，其独特地适合于解决MDMA诱导的损伤的假设机制。这些实验将直接测量MDMA、酪氨酸、高热和细胞内多巴胺的形成对5-HT神经元内氧化过程的个体和相对影响，以及这些变量如何影响细胞活力。结合体内微透析研究，该模型的测试提供了一种独特而强大的方法来解决先前与MDMA诱导的5 HT系统神经变性相关的神秘问题

DESCRIPTION (provided by applicant): The widely abused amphetamine analogue, 3 4-methylenedioxymethamphetamine (MDMA, Ecstasy) selectively damages the axon terminals of 5-HT neurons in the brain. Several lines of evidence suggest that dopamine (DA) contributes to this toxicity. Although DA clearly produces oxidative by-products such as reactive oxygen species and quinones, it remains unclear as to how DA-derived oxidative species produce selective damage to 5HT terminals, a hallmark of MDMA-induced neurotoxicity. More specifically, the mechanism as to how DA accumulates within 5-HT terminals to produce its selective damage is unknown. The overaraching hypothesis of the current proposal is that L- tyrosine, the amino acid precursor of DA, contributes to the neurodegenerative process. This hypothesis is based on our recent preliminary data indicating a 5-fold increase in the extracellular concentration of tyrosine measured in vivo after MDMA. Unlike DA, tyrosine is actively transported from the periphery and into the brain and neurons. While tyrosine is the natural precursor for DA synthesis within DA neurons, high concentrations of tyrosine in 5HT neurons may have deleterious consequences. The hypothetical framework of this proposal is that the oxidative environment produced by MDMA and hyperthermia in 5-HT neurons causes the non-enzymatic oxidation of tyrosine to the DA precursor, DOPA. Aromatic amino acid decarboxylase (AADC), within the 5-HT terminal then would decarboxylate DOPA to DA, leading to an accumulation of DA and consequently, DA-derived reactive oxygen species and oxidative damage within 5HT terminals. This hypothesized role of tyrosine as a mediator of MDMA-induced toxicity is a novel mechanism that effectively synthesizes current existing hypotheses and to some extent, discrepant observations and apparent caveats into a cohesive, theoretical and testable framework. The Specific Aims are (1) to demonstrate the non-enzymatic oxidation of tyrosine to DOPA and DA within 5HT neurons, (2) to characterize the contributions of L-tyrosine and tyramine to MDMA and hyperthermia-induced oxidative stress and (3) to assess subsequent neuronal damage. The use of cultured RN46A 5HT cells is a novel approach that is uniquely suited to address the hypothesized mechanism of MDMA-induced damage. These experiments will directly measure the individual and relative effects of MDMA, tyrosine, hyperthermia, and the formation of intracellular dopamine on oxidative processes within 5-HT neurons and how these variables affect cell viability. The testing of this model in conjunction with in vivo microdialysis studies, provides a unique and powerful approach to address enigmatic issues previously related to MDMA-induced neurodegeneration of 5HT systems