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）暴露会损伤脑组织;然而，尚未完全了解其潜在机制。基于我们最近的工作，即新发现的细胞死亡介质转化生长因子-可诱导早期基因2（TIEG 2）蛋白在人脑细胞和成年大鼠大脑中被酒精显著增加，这项创新建议的目的是进一步表征TIEG 2在EtOH诱导的脑损伤中的作用。 TIEG 2是抑制细胞生长、诱导细胞凋亡和增加单胺氧化酶B（MAO B）表达的转录因子。MAO B的酶活性产生H2 O2，这是活性氧（ROS）毒性的主要原因。据报道，EtOH增加了MAO B的活性，并且其次增加了H2 O2的产生。我们发表的数据表明，生理相关的EtOH增加了神经元细胞系中TIEG 2-MAO B途径的表达。TIEG 2的过度表达增强了乙醇诱导的神经元死亡，而MAO B的抑制剂减少了乙醇诱导的神经元死亡。此外，常见的TIEG 2基因变体（Gln 62 Arg，TIEG 2的多态性）改变了TIEG 2的活性，并使细胞对氧化应激比TIEG 2野生型更敏感。因此，我们假设乙醇诱导TIEG 2及其变体（MAO B转录激活因子）的表达。其次，我们假设，MAO B的抑制剂可以通过减少TIEG 2-MAO B产生的活性氧（ROS）来提供对乙醇诱导的脑组织损伤的保护。我们的具体目的是：（1）鉴定乙醇诱导的TIEG 2上调中的分子信号成分;（2）确定TIEG 2基因变异体是否与乙醇诱导的TIEG 2上调有关。（3）确定MAO B抑制剂对乙醇诱导的细胞毒性的保护作用;（4）观察单胺氧化酶（MAO）B抑制剂对乙醇所致大鼠脑组织神经毒性的保护作用。 分别通过定量实时RT-PCR、蛋白质印迹和TUNEL测定法测定TIEG 2、MAO B和细胞死亡标志物的水平。还将测定细胞增殖速率、ROS的产生和神经变性。将在不同的组之间进行比较：未处理的对照组;乙醇处理组; MAO B抑制剂处理组;以及伴随有MAO B抑制剂的乙醇处理组。 我们的提案将研究涉及TIEG 2和MAO B在乙醇诱导的神经毒性中的新途径的潜在作用，并确定可能赋予乙醇诱导的脑细胞损伤易感性的遗传风险因子。它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转化研究。因此，该提案有可能对公共卫生产生重大影响。

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.