Ethanol induced brain injury is decreased by inhibiting TIEG2 mediated cell death

描述(由申请人提供)：众所周知，乙醇(Etoh)暴露会损害脑组织；然而，其潜在的机制还不完全清楚。在我们最近工作的基础上，我们最近发现的细胞死亡介质，转化生长因子诱导的早期基因2(TIEG2)蛋白，在酒精诱导的人脑细胞和成年大鼠脑中显著增加，这项创新建议的目标是进一步表征TIEG2在乙醇诱导的脑损伤中的作用。TIEG2是一种转录因子，可以抑制细胞生长，诱导细胞凋亡，并增加单胺氧化酶B(MAO B)的表达。MAO B的酶活性会产生过氧化氢，这是导致活性氧物种(ROS)毒性的主要原因。据报道，乙醇增加了MAO B的活性，继而增加了过氧化氢的产生。我们发表的数据表明，生理上相关的乙醇增加了神经细胞系中TIEG2-MAO B通路的表达。TIEG2的过度表达增强，而MAO B的抑制剂减少乙醇诱导的神经元死亡。此外，频繁的TIEG2基因变异(Gln62Arg，TIEG2的一个多态)改变了TIEG2的活性，使细胞对氧化应激比TIEG2野生型更敏感。因此，我们假设乙醇诱导TIEG2及其变异体(MAO B转录激活子)的表达。其次，我们假设MAO B的抑制剂可能通过减少TIEG2-MAO B产生的活性氧(ROS)而对乙醇诱导的脑组织损伤起到保护作用。我们的具体目标是(1)确定与乙醇诱导的TIEG2上调有关的分子信号成分；(2)确定TIEG2基因变体(Gln62Arg)是否比TIEG2野生型更敏感于乙醇毒性；(3)确定MAO B抑制剂对乙醇诱导的细胞存活的保护作用；以及(4)检测MAO B抑制剂对乙醇诱导的成年大鼠脑组织神经毒性的保护作用。TIEG2、MAO B和细胞死亡标记物的水平将分别通过实时定量RT-PCR、Western印迹和TUNEL检测。细胞增殖率、ROS的产生和神经变性也将被测定。比较不同组之间的差异：未处理的对照组；乙醇处理组；MAO B抑制剂处理组；乙醇联合MAO B抑制剂处理组。我们的建议将研究涉及TIEG2和MAO B的新通路在乙醇诱导的神经毒性中的潜在作用，并确定可能使人对乙醇诱导的脑细胞损伤易感性的遗传风险因素。这也将作为开发新的抗氧化剂治疗乙醇诱导的脑组织损伤的翻译研究。因此，这项提议有可能极大地影响公众健康。

DESCRIPTION (provided by applicant): It is well known that ethanol (EtOH) exposure damages brain tissue; however, the underlying mechanisms are not fully understood. Building on our recent work that the newly discovered cell death- mediator, transforming growth factor-¿-inducible early gene 2 (TIEG2) protein, is significantly increased by alcohol in human brain cells and also in adult rat brains, the objective of this innovative proposal is to further characterize the role of TIEG2 in EtOH-induced brain damage. TIEG2 is a transcription factor that inhibits cell growth, induces apoptosis, and increases the expression of monoamine oxidase B (MAO B). The enzymatic activity of MAO B generates H2O2, a major cause of reactive oxygen species (ROS) toxicity. EtOH reportedly increases the activity of MAO B, and secondarily increases the production of H2O2. Our published data show that physiologically-relevant EtOH increases the expression of the TIEG2-MAO B pathway in a neuronal cell line. Over-expression of TIEG2 enhances, whereas inhibitors of MAO B reduce EtOH-induced neuronal death. Additionally, a frequent TIEG2 gene variant (Gln62Arg, a polymorphism of TIEG2) alters the activity of TIEG2 and renders cells more sensitive to oxidative stress than the TIEG2 wild type. Therefore, we hypothesize that ethanol induces the expression of TIEG2 and its variant (the MAO B transcriptional activators). Secondly, we hypothesize that inhibitors of MAO B may provide protection against ethanol-induced brain tissue injury by reducing the TIEG2-MAO B- produced reactive oxygen species (ROS). Our Specific Aims are (1) To identify the molecular signaling components involved in ethanol-induced up-regulation of TIEG2; (2) To determine whether the TIEG2 gene variant (Gln62Arg) sensitizes cells to ethanol toxicity more than the TIEG2 wild type; (3) To determine the protective effects of MAO B inhibitors on cellular survival against ethanol-induced toxicity; and (4) To examine the protective effects of MAO B inhibitors on ethanol-induced neurotoxicity in adult rat brain tissues. The levels of TIEG2, MAO B, and cell death markers will be determined by quantitative real-time RT- PCR, Western blot and TUNEL assays, respectively. The cell proliferation rate, the production of ROS, and the neurodegeneration will also be determined. A comparison will be made among different groups: untreated controls; ethanol-treated; MAO B inhibitor-treated; and ethanol-treated accompanied with MAO B inhibitors. Our proposal will examine the potential role of a novel pathway involving TIEG2 and MAO B in EtOH- induced neurotoxicity and identifies a genetic risk factor that may confer susceptibility to ethanol-induced brain cell damage. It will also serve as the translational study for developing new antioxidant therapeutics for ethanol-induced brain tissue injury. Therefore, this proposal has the potential to greatly impact public health.