Apoptotic Mechanism of Maternal Diabetes-Induced Neural Tube Defects

描述（由申请人提供）：1型或2型糖尿病妇女所生的婴儿中，严重先天性畸形发生率高达10%，导致严重的公共卫生问题。母体高血糖期间的先天性畸形是靶组织过度凋亡的结果。母体高血糖激活促凋亡级联反应，导致胚胎细胞过度凋亡，导致胚胎畸形发生。我们发表的关于caspase 8和JNK2的数据，Foxo3a缺乏改善糖尿病胚胎病的初步数据，以及植物化学物质EGCG通过抑制Foxo3a激活对高血糖诱导的畸形的预防作用，都暗示了Foxo3a中心的促凋亡级联反应在该疾病过程中。我们假设JNK1/2、Foxo3a、TRADD、caspase 8通路可促进细胞凋亡，而Foxo3a是TRADD转录的关键激活因子。TRADD随后诱导发育中的胚胎神经上皮细胞凋亡，导致该疾病的特征神经管缺陷（神经管缺陷，NTD）（图1）。植物化学物质EGCG通过阻断这一途径减少糖尿病诱导的NTD。在Specific Aim 1中，我们将确定Foxo3a是否是JNK1/2在导致高血糖诱导的胚胎畸形通路中的一个关键下游靶点。我们将详细剖析糖尿病诱导Foxo3a激活与JNK相关的机制。我们将在JNK2缺乏的情况下监测细胞质/细胞核Foxo3a磷酸化状态、Foxo3a与14-3-3相互作用、核易位和DNA结合。我们将确定Foxo3a在高血糖诱导的细胞凋亡和胚胎畸形中是否必需（目的2）。我们假设Foxo3a活性是激活TRADD表达所必需的。我们将使用Foxo3a敲除（Foxo3aKO）小鼠来检测TRADD表达、caspase依赖性细胞凋亡和胚胎畸形是否需要Foxo3a。我们将确定TRADD在母体高血糖诱导的胚胎病中是否需要细胞凋亡，以及EGCG在糖尿病胚胎病和糖尿病诱导的促凋亡通路中的作用（目的3）。trad - fadd复合物触发caspase 8激活导致细胞凋亡。我们将使用？-actin-FADD-DN （Dominant Negative）转基因小鼠，检测阻断TRADD功能是否能阻止高血糖诱导的畸形、caspase 8激活和细胞凋亡。使用非糖尿病和糖尿病妊娠小鼠，我们将通过给予膳食EGCG补充剂来确定EGCG在体内的作用。我们将确定EGCG对母体糖尿病诱导的NTD、JNK1/2和Foxo3a磷酸化、Foxo3a核易位、TRADD上调、caspase 8切割和凋亡的影响。Caspase 8是糖尿病胚胎病中发现的凋亡启动因子之一，其激活可导致Bcl-2家族成员和效应Caspase 3的激活。我们进一步定义了转录因子和凋亡基因介导糖尿病诱导的caspase 8激活和凋亡，以Bcl-2和caspase 3为终点。在如此复杂的疾病中使用优雅的转基因小鼠模型将在该领域产生重大影响。为了研究EGCG的作用，我们将我们的机制研究与一种可能的治疗候选药物联系起来。我们的研究和方法的创新包括Foxo3a在其他Foxo因子中的关键作用，潜在的翻译EGCG研究，精心设计的转基因小鼠使用，以及首次定义糖尿病诱导Foxo3a激活的详细机制

DESCRIPTION (provided by applicant): Major congenital malformations occur in up to 10% of babies born to women with type 1 or 2 diabetes resulting in a significant public health problem. Congenital malformations during maternal hyperglycemia are the result of excess apoptosis in target tissues. Maternal hyperglycemia activates pro-apoptotic cascades resulting in excess apoptosis in embryonic cells leading to embryonic dysmorphogenesis. Our published data in caspase 8 and JNK2, preliminary data in amelioration of diabetic embryopathy by Foxo3a deficiency and preventive effect of phytochemical EGCG on hyperglycemia-induced malformations via inhibition of Foxo3a activation, implicate a Foxo3a centric pro-apoptotic cascade in this disease process. We hypothesize that a JNK1/2, Foxo3a, TRADD, caspase 8 pathway acts to enhance apoptosis, and that Foxo3a is a key activator of TRADD transcription. TRADD then induces apoptosis in the neuroepithelium of the developing embryo leading to neural tube defects (Neural tube defect, NTD) characteristic of the disease (Fig. 1). Phytochemical EGCG reduces diabetes-induced NTD via blockade of this pathway. In Specific Aim 1, we will determine if Foxo3a is a key downstream target of JNK1/2 in the pathway leading to hyperglycemia-induced embryonic malformation. We will dissect the detailed mechanisms whereby diabetes-induced Foxo3a activation in connection with JNK. We will monitor cytoplasmic/nuclear Foxo3a phosphorylation state, Foxo3a and 14-3-3 interaction, nuclear translocation and DNA binding under JNK2 deficiency. We will determine if Foxo3a is required for hyperglycemia-induced apoptosis and embryonic malformation (Aim 2). We hypothesize that Foxo3a activity is required for activation of TRADD expression. We will use Foxo3a knockout (Foxo3aKO) mice to test whether Foxo3a is required for TRADD expression, caspase-dependent apoptosis, and embryo malformation. We will determine if TRADD is required for apoptosis in maternal hyperglycemia-induced embryopathy, and the effect of EGCG on diabetic embryopathy and the diabetes-induced pro-apoptotic pathway (Aim 3). TRADD-FADD complex triggers caspase 8 activation leading to apoptosis. We will use ?-actin-FADD-DN (Dominant Negative) transgenic mice to test whether blockade of TRADD function prevents hyperglycemia-induced malformation, caspase 8 activation and apoptosis. Using non-diabetic and diabetic pregnant mice, we will determine EGCG's effects in vivo by administering dietary EGCG supplements. We will determine EGCG's effects on maternal diabetesinduced NTD, phosphorylation of JNK1/2 and Foxo3a, Foxo3a nuclear translocation, upregulation of TRADD, caspase 8 cleavage and apoptosis. Caspase 8 is one of the apoptosis initiator being identified in diabetic embryopathy and its activation leads to activation of Bcl-2 family members and effector caspases such as caspase 3. We further define the transcription factor and the apoptotic gene mediating diabetes-induced caspase 8 activation and apoptosis using our previous findings in Bcl-2 and caspase 3 as endpoints. Using elegant genetically modified mouse models in such a complex disease would have high impact in this field. To study the effect of EGCG, we bridge our mechanistic studies to a possible therapeutic candidate. The innovations of our studies and approaches include the critical role of Foxo3a among other Foxo factors, potential translational EGCG studies, well-designed use of genetically modified mice and first defining the detailed mechanisms whereby diabetes-induced Foxo3a activation