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，其潜在机制尚未完全了解。基于我们最近的工作，新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，在人类脑细胞和成年大鼠大脑中被酒精显著增加，这一创新建议的目的是进一步表征TIEG2在乙醇诱导的脑损伤中的作用。TIEG2是一种抑制细胞生长、诱导细胞凋亡、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H2O2，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，进而增加了H2O2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TIEG2-MAO B通路的表达。TIEG2的过表达增强，而MAO B的抑制剂可减少etoh诱导的神经元死亡。此外，一种常见的TIEG2基因变异（Gln62Arg， TIEG2的多态性）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的抑制剂可能通过减少TIEG2-MAO B产生的活性氧（ROS）来保护乙醇诱导的脑组织损伤。我们的具体目标是：(1)确定参与乙醇诱导的TIEG2上调的分子信号成分；(2)确定TIEG2基因变异（Gln62Arg）是否比TIEG2野生型更能使细胞对乙醇毒性敏感；(3)确定MAO B抑制剂对乙醇毒性小鼠细胞存活的保护作用；(4)观察MAO B抑制剂对乙醇致成年大鼠脑组织神经毒性的保护作用。TIEG2、MAO B和细胞死亡标志物的水平将分别通过定量实时RT- PCR、Western blot和TUNEL测定。细胞增殖率、ROS的产生和神经退行性变也将被确定。将在不同组之间进行比较：未经治疗的对照组；ethanol-treated;MAO B抑制剂处理；乙醇处理和MAO B抑制剂。我们的提案将研究涉及TIEG2和MAO B的新途径在EtOH诱导的神经毒性中的潜在作用，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素。它也将为开发新的抗氧化治疗乙醇性脑组织损伤的转化研究。因此，这一建议有可能对公众健康产生重大影响。

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.