Apoptotic Mechanism of Maternal Diabetes-Induced Neural Tube Defects

描述(由申请人提供)：患有1型或2型糖尿病的妇女所生的婴儿中，高达10%会发生严重的先天性畸形，导致严重的公共卫生问题。母体高血糖时的先天性畸形是靶组织细胞过度凋亡的结果。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。我们在caspase 8和JNK2上发表的数据，关于Foxo3a缺乏改善糖尿病胚胎病变的初步数据，以及植物化学物质EGCG通过抑制Foxo3a激活预防高血糖诱导的畸形，暗示Foxo3a在这一疾病过程中存在以Foxo3a为中心的促凋亡级联反应。我们推测JNK1/2、Foxo3a、Tradd、caspase 8途径促进细胞凋亡，Foxo3a是Tradd转录的关键激活因子。然后，TRADD诱导发育中胚胎的神经上皮细胞凋亡，导致神经管缺陷(神经管缺陷，NTD)，这是这种疾病的特征(图1)。植物化学成分EGCG通过阻断这一途径减少糖尿病引起的NTD。在特定的目标1中，我们将确定Foxo3a是否是JNK1/2在导致高血糖诱导胚胎畸形的途径中的关键下游靶点。我们将剖析糖尿病诱导的Foxo3a激活与JNK相关的详细机制。我们将监测JNK2缺乏时细胞质/核Foxo3a的磷酸化状态、Foxo3a与14-3-3的相互作用、核转位和DNA结合。我们将确定Foxo3a是否是高血糖诱导的细胞凋亡和胚胎畸形所必需的(目标2)。我们假设，激活Tradd表达需要Foxo3a活性。我们将使用Foxo3a基因敲除(Foxo3aKO)小鼠来测试Foxo3a是否是Tradd表达、caspase依赖的细胞凋亡和胚胎畸形所必需的。我们将确定TRADD是否是母亲高血糖诱导的胚胎病变中的细胞凋亡所必需的，以及EGCG在糖尿病胚胎病变和糖尿病诱导的促细胞凋亡途径中的作用(目标3)。Tradd-FADD复合体触发caspase 8激活，导致细胞凋亡。我们将使用？-肌动蛋白-FADD-dN(显性阴性)转基因小鼠来测试阻断Tradd功能是否可以防止高血糖诱导的畸形、caspase8激活和细胞凋亡。使用非糖尿病和糖尿病怀孕的小鼠，我们将通过补充饮食中的EGCG来确定EGCG在体内的影响。我们将检测EGCG对母体糖尿病所致NTD、JNK1/2和Foxo3a的磷酸化、Foxo3a核转位、Tradd上调、caspase8裂解和细胞凋亡的影响。Caspase8是糖尿病胚胎病变中发现的一种细胞凋亡启动子，它的激活导致Bcl2家族成员和效应Caspase如Caspase3的激活。我们结合我们以前在Bcl2和Caspase3中的发现，进一步定义了介导糖尿病诱导的caspase8激活和细胞凋亡的转录因子和凋亡基因。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很大影响。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EGCG翻译研究，精心设计的转基因小鼠的使用，以及首次确定糖尿病诱导Foxo3a激活的详细机制 与公共卫生相关：患有1型或2型糖尿病的妇女所生的婴儿中，有高达10%的婴儿会发生严重的先天性畸形，如神经管缺陷，从而造成严重的公共卫生问题。本研究旨在鉴定导致糖尿病胚胎病变的细胞凋亡中间产物，并从细胞水平和转录水平确定糖尿病胚胎病变的发病机制。通过揭示导致糖尿病胚胎病变的机制，这项研究的结果将为使用尖端的、基于机制的治疗策略提供机制基础，旨在预防糖尿病相关的出生缺陷。

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 PUBLIC HEALTH RELEVANCE: Major congenital malformations such as neural tube defects occur in up to 10% of babies born to women with type 1 or 2 diabetes resulting in a significant public health problem. The proposed study is to identify apoptotic intermediates responsible for the induction of diabetic embryopathy and define the mechanism of diabetic embryopathy at both the cellular and transcriptional levels. By unraveling the mechanisms leading to diabetic embryopathy, the results of the study will provide a mechanistic basis for the use of cutting-edge, mechanism-based therapeutic strategies designed to prevent diabetes-associated birth defects.