Role of Tyrosine in MDMA Toxicity

描述(申请人提供)：被广泛滥用的苯丙胺类似物，3，4-亚甲基二氧基甲基苯丙胺(MDMA，摇头丸)选择性地损害大脑中5-羟色胺神经元的轴突终末。一些证据表明，多巴胺(DA)导致了这种毒性。尽管DA明显产生氧化副产物，如活性氧和苯二酚，但DA衍生的氧化物种如何对5-羟色胺终末产生选择性损伤仍不清楚，这是MDMA诱导的神经毒性的标志。更具体地说，DA如何在5-羟色胺终末内蓄积从而产生选择性损伤的机制尚不清楚。目前的假说认为，多巴胺的氨基酸前体L酪氨酸参与了神经退行性变过程。这一假设是基于我们最近的初步数据，这些数据表明MDMA后体内测得的细胞外酪氨酸浓度增加了5倍。与DA不同的是，酪氨酸从外周主动运输到大脑和神经元。虽然酪氨酸是DA神经元合成DA的天然前体，但5HT神经元中高浓度的酪氨酸可能会产生有害的后果。这一建议的假设框架是，MDMA和高温在5-羟色胺神经元中产生的氧化环境导致酪氨酸非酶氧化为DA前体DOPA。5-羟色胺末端的芳香族氨基酸脱羧酶(AADC)会将DOPA脱羧为DA，导致DA的积累，从而导致DA衍生的活性氧物种和5HT末端的氧化损伤。酪氨酸作为MDMA诱导毒性的媒介这一假设作用是一种新的机制，它有效地综合了现有的假设，在某种程度上，不一致的观察和明显的警告进入了一个连贯的、理论的和可测试的框架。 其具体目的是(1)证明5HT神经元内酪氨酸非酶氧化为DOPA和DA，(2)表征L酪氨酸和酪胺在高温诱导的氧化应激和MDMA中的作用，以及(3)评估随后的神经元损伤。使用培养的RN46A 5HT细胞是一种新的方法，特别适合于解决MDMA诱导损伤的假设机制。这些实验将直接测量MDMA、酪氨酸、高温和细胞内多巴胺的形成对5-羟色胺神经元内氧化过程的个别和相对影响，以及这些变量如何影响细胞活力。这一模型的测试与体内微透析研究相结合，提供了一种独特而强大的方法来解决以前与MDMA诱导的5HT系统神经变性有关的谜团问题

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