Apoptotic Mechanism of Maternal Diabetes-Induced Neural Tube Defects

描述（由申请人提供）：1型或2型糖尿病女性所生婴儿中，高达10%的婴儿发生严重的先天性畸形，导致严重的公共卫生问题。母体高血糖期间的先天性畸形是靶组织中过量凋亡的结果。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。我们发表的关于半胱天冬酶8和JNK 2的数据，Foxo 3a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Foxo 3a活化对高血糖诱导的畸形的预防作用，暗示了Foxo 3a中心的促凋亡级联反应在这种疾病过程中。我们推测JNK 1/2、Foxo 3a、TRADD、caspase 8通路可增强细胞凋亡，Foxo 3a是TRADD转录的关键激活因子。然后TRADD诱导发育胚胎神经上皮细胞凋亡，导致疾病特征性神经管缺陷（神经管缺陷，NTD）（图1）。植物化学物质EGCG通过阻断该途径减少糖尿病诱导的NTD。在具体目标1中，我们将确定Foxo 3a是否是导致高血糖诱导的胚胎畸形的通路中JNK 1/2的关键下游靶标。我们将详细分析糖尿病诱导Foxo 3a激活与JNK相关的机制。我们将监测JNK 2缺陷下细胞质/核Foxo 3a磷酸化状态、Foxo 3a和14-3-3相互作用、核转位和DNA结合。我们将确定Foxo 3a是否是高血糖诱导的细胞凋亡和胚胎畸形所必需的（目的2）。我们假设Foxo 3a活性是TRADD表达激活所必需的。我们将使用Foxo 3a敲除（Foxo 3aKO）小鼠来测试Foxo 3a是否是TRADD表达、半胱天冬酶依赖性凋亡和胚胎畸形所必需的。我们将确定TRADD是否是母体高血糖诱导的胚胎病中细胞凋亡所必需的，以及EGCG对糖尿病胚胎病和糖尿病诱导的促细胞凋亡途径的影响（目的3）。TRADD-FADD复合物触发半胱天冬酶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，以测试TRADD功能的阻断是否防止高血糖诱导的畸形、半胱天冬酶8活化和细胞凋亡。使用非糖尿病和糖尿病妊娠小鼠，我们将通过给予膳食补充剂来确定EGCG在体内的作用。我们将研究EGCG对糖尿病诱导的NTD、JNK 1/2和Foxo 3a磷酸化、Foxo 3a核转位、TRADD上调、caspase 8裂解和细胞凋亡的影响。半胱天冬酶8是在糖尿病胚胎病中鉴定的细胞凋亡起始物之一，其活化导致Bcl-2家族成员和效应半胱天冬酶如半胱天冬酶3的活化。我们进一步定义了转录因子和凋亡基因介导的糖尿病诱导的caspase 8激活和凋亡，使用我们以前的研究结果在Bcl-2和caspase 3作为终点。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巨大影响。为了研究EGCG的作用，我们将我们的机制研究与可能的治疗候选者联系起来。我们的研究和方法的创新包括Foxo 3a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的使用，以及首次定义糖尿病诱导Foxo 3a激活的详细机制 公共卫生相关性：患有1型或2型糖尿病的妇女所生的婴儿中，高达10%的婴儿患有神经管缺陷等重大先天性畸形，导致严重的公共卫生问题。本研究拟在细胞和转录水平上鉴定诱导糖尿病胚胎病的凋亡中间体，并确定糖尿病胚胎病的发病机制。通过揭示导致糖尿病胚胎病的机制，该研究的结果将为使用旨在预防糖尿病相关出生缺陷的尖端、基于机制的治疗策略提供机制基础。

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.