Intersection of the mTOR/p70S6K1 signaling and the HIPPO-Yap tissue organizer in neurulation and diabetic embryopathy

摘要 妊娠期糖尿病可导致神经管畸形(NTDS)。有6000万育龄妇女 (18-44岁)，全世界约有300万美国妇女患有糖尿病。即使在最好的情况下 产前护理，糖尿病妇女生下有出生缺陷的孩子的可能性仍然是 没有糖尿病的女性。解开糖尿病引起的NTDS的潜在机制对于 了解其发病机制，并提供潜在的干预靶点。是相关蛋白(YAP)， 转录共激活因子，决定身体和器官的大小，并调节细胞的凋亡、增殖和 差异化。我们发现，无论是YAP的条件性KO还是转基因(TG)的结构性过表达 神经上皮细胞中的活性YAP(CA-YAP)可导致NTD的形成。我们最近公布的数据 证明mTOR(雷帕霉素的机械靶点)下游的一个激酶p70S6K1被激活 通过母体糖尿病及其缺乏症缓解糖尿病胚胎病变。此外，mTOR抑制剂 雷帕霉素可预防糖尿病胚胎病变。河马-YAP通路和mTOR通路均诱导细胞 和组织生长。因此，我们假设母亲糖尿病激活了mTOR-p70S6K1，这是 通过磷酸化激活河马激酶Lats1来对抗YAP，选择性增加 翻译并破坏内质网(ER)的动态平衡，导致NTDS。Lats1停用 YAP通过磷酸化，破坏平面细胞极性(PCP)，导致失败的神经分化。至 为了验证我们的假设，我们提出了以下目标。具体目标1.确定主要mTOR是否 下游效应子p70S6K1通过磷酸化YAP上游激酶Lats1来抵消YAP活性 糖尿病胚胎病变。我们将研究母亲糖尿病是否激活了mTOR效应器p70S6K1， 它与支架蛋白Merlin和Lats1形成三级复合体，在两个阶段触发Lats1磷酸化 之前未识别的位点Thr255和Thr262导致YAP失活。具体目标2.确定 河马信号通路Lats1激活是否导致糖尿病大鼠神经管畸形 胚胎病。我们将测试母体糖尿病是否会导致Lats1在新的位点和 随后是导致抑制YAP和NTD形成的典型的磷酸化位点。特定的 目的3.研究微调的YAP活动如何对神经形成至关重要，以及它的失调如何扰乱 糖尿病胚胎病变中的平面细胞极化。我们将评估YAP活动在 神经再生和恢复YAP活性可改善糖尿病胚胎病变。我们将进一步评估是否 微调的YAP活性在神经形成过程中维持PCP基因的表达。

SUMMARY Pregestational diabetes induces neural tube defects (NTDs). There are 60 million women of reproductive age (18-44 years old) worldwide, and approximately 3 million American women with diabetes. Even under the best prenatal care, women with diabetes are still three- to four-times more likely to have a child with birth defects than women without diabetes. Unraveling the mechanism underlying diabetes-induced NTDs is critical for understanding its pathogenesis and providing potential intervention targets. Yes-associated protein (Yap), a transcriptional co-activator, determines body and organ size and regulates cell apoptosis, proliferation, and differentiation. We found that either conditional KO of Yap or transgenic (Tg) overexpression of constitutively active Yap (CA-Yap) specifically in the neuroepithelium resulted in NTD formation. Our recently published data demonstrate that one of the mTOR (mechanistic target of rapamycin) downstream kinase p70S6K1 is activated by maternal diabetes and its deficiency alleviates diabetic embryopathy. Furthermore, the mTOR inhibitor rapamycin prevents diabetic embryopathy. Both the HIPPO-Yap pathway and the mTOR pathway induce cell and tissue growth. Thus, we hypothesize that maternal diabetes activates mTOR-p70S6K1, which antagonizes Yap by activating the HIPPO kinase Lats1 through phosphorylation, selectively increases translation and disrupts endoplasmic reticulum (ER) homeostasis, leading to NTDs. Lats1 inactivates Yap through phosphorylation that disrupts the planar cell polarity (PCP) leading to failed neurulation. To test our hypothesis, we propose the following aims. Specific Aim 1. determine whether the major mTOR downstream effector p70S6K1 counteracts Yap activity by phosphorylating Yap upstream kinase Lats1 in diabetic embryopathy. We will examine whether maternal diabetes-activated the mTOR effector p70S6K1, which forms a tertiary complex with the scaffold protein Merlin and Lats1, triggers Lats1 phosphorylation in two previously unidentified sites, Thr255 and Thr262, leading to Yap inactivation. Specific Aim 2. To determine whether the HIPPO signaling kinase Lats1 activation leads to neural tube defects in diabetic embryopathy. We will test whether maternal diabetes induces Lats1 phosphorylation at new sites and subsequent the canonical phosphorylation sites which leads to suppression of Yap and NTD formation. Specific Aim 3. To investigate how fine-tuned Yap activity is critical for neurulation and its dysregulation disrupts planar cell polarity in diabetic embryopathy. We will assess that Yap activity is tightly controlled during neurulation and restoring Yap activity can ameliorate diabetic embryopathy. We will further evaluate whether fine-tuned Yap activity maintains PCP gene expression during neurulation.