Transglutaminase 2 decreases ischemic insult by attenuating hypoxic signaling.

描述（由申请人提供）：中风是美国第三大死亡原因，也是长期残疾的主要原因。目前溶栓治疗的不充分成功表明迫切需要新的靶点来改善卒中后生活质量。神经元通过上调由缺氧诱导因子（HIF）控制的基因来适应中风后氧气输送的减少。该转录因子由两个亚基组成：HIF 1 α和HIF 1 β。最近的研究表明，在缺血期间增加HIF 1导致凋亡基因的上调。我们的初步数据表明，转氨酶2（TG 2）结合HIF 1 β，导致缺血期间HIF激活减弱和细胞死亡减少。此外，TG 2抑制HIF控制的促凋亡基因Bnip 3的上调，但不抑制VEGF。我们的假设是，TG 2将促进神经元保护后，氧和葡萄糖剥夺（OGD）通过衰减HIF 1诱导的凋亡基因。我们提出描绘TG 2对HIF信号传导和缺血性细胞死亡的影响，有3个目的。1)为了检验TG 2在OGD期间上调并降低HIF依赖性促凋亡信号传导的假设。我们将使用过表达野生型TG 2的神经母细胞瘤细胞系（SH-SY 5 Y）和两种具有点突变的TG 2构建体，这些点突变差异性地影响酶的2种独特功能（转酰胺和GTdR）。使用HIF信号报告分析和HIF依赖性基因的mRNA和蛋白质表达模式的分析，我们将区分TG 2的哪些特性对于调节HIF信号传导是重要的。2)为了验证TG 2保护神经细胞免受缺血损伤的假设以及TG 2的功能是重要的。使用SH-SY 5 Y细胞和原代皮层神经元，我们将测量OGD诱导的细胞死亡和上游凋亡事件。3)在小鼠中风模型中检验TG 2导致总体保护的假设。我们将在表达内源性TG 2、过表达TG 2或缺乏TG 2的C57 BL/6小鼠中使用永久性大脑中动脉结扎模型。我们将使用T2加权MRI测量梗死体积，并使用适当的技术识别由TG 2调控的基因。我们的初步数据显示，TG 2的过度表达可使卒中梗死体积减少33%。我们假设TG 2将导致对中风的全面保护。

DESCRIPTION (provided by applicant): Stroke is the third leading cause of death and a major cause of long term disability in the US. Inadequate success of current thrombolytic therapy demonstrates a critical need for new targets to improve quality of life post-stroke. Neurons adapt to decreased oxygen delivery subsequent to stroke by up-regulating genes controlled by the hypoxia inducible factor (HIF). This transcription factor is comprised of two subunits: HIF1 alpha and HIF1 beta. Recent studies show that increased HIF1 during ischemia results in upregulation of apoptotic genes. Our preliminary data indicates that Transglutaminase 2 (TG2) binds to HIF1 beta resulting in attenuation of HIF activation and decreased cell death during ischemia. Also, TG2 inhibits the upregulation of the HIF controlled proapoptotic gene Bnip3, but not VEGF. Our hypothesis is that TG2 will facilitate neuronal protection following oxygen and glucose deprivation (OGD) by attenuating apoptotic genes induced by HIF1. We propose to delineate the effects of TG2 on HIF signaling and ischemic cell death with 3 aims. 1) To test the hypothesis that TG2 is upregulated during OGD and decreases HIF-dependent pro-apoptotic signaling. We will use a neuroblastoma cell line (SH-SY5Y) that overexpresses wild type TG2 and two TG2 constructs with point mutations that differentially affect 2 unique functions (transamidating and GTPase) of the enzyme. Using a HIF signaling reporter assay and analysis of mRNA and protein expression patterns of HIF dependent genes, we will distinguish which properties of TG2 are important for regulating HIF signaling. 2) To test the hypothesis that TG2 protects neuronal cells from ischemic insult and which functions of TG2 are important. Using SH-SY5Y cells and primary cortical neurons we will measure OGD-induced cell death and upstream apoptotic events. 3) To test the hypothesis that TG2 leads to overall protection in a mouse model of stroke. We will use a permanent Middle Cerebral Artery ligation model in C57BL/6 mice that express endogenous TG2, overexpress TG2, or lack TG2. We will measure infarct volumes using T2 weighted MRI as well as identifying genes regulated by TG2 with appropriate techniques. Our preliminary data show that overexpression of TG2 decreases stroke infarct volumes by 33%. We hypothesize that TG2 will lead to overall protection against stroke.