Autophagy and its Regulation in Diabetic Embryopathy

描述（由申请人提供）：先天性畸形发生在高达10%的婴儿出生的糖尿病妇女。最佳血糖控制难以实现和维持，即使短暂暴露于高血糖也会导致畸形。该项目是在我们强大的初步数据的基础上制定的。我们发现1）母体糖尿病损害自噬并增加发育中神经上皮中缺陷线粒体、功能障碍蛋白和肿胀内质网（ER）的积累; 2）无毒自噬激活剂海藻糖逆转糖尿病诱导的自噬损害和神经管缺陷（NTDs）; 3）异硫氰酸荧光素（FITC）标记的海藻糖与自噬促进因子Beclin-1和ATG 12结合，诱导选择性自噬; 4）PKCa基因缺失、p70 S6 K1抑制剂和自噬促进因子AMBRA 1在神经管中的过表达均减少糖尿病诱导的NTD。我们测试了一种新的假设，即海藻糖通过重新组装糖尿病破坏的自噬起始复合物来激活自噬，消除p70 S6 K1的抑制并恢复AMBRA 1表达。神经上皮中S6 K1基因的缺失和AMBRA 1基因的过表达都能重新激活自噬并恢复细胞内稳态，从而预防NTD。目的1将确定海藻糖诱导选择性自噬和恢复细胞内稳态从而预防糖尿病胚胎病的潜在机制。我们假设海藻糖通过促进PI 3 KC 3-Beclin-1-AMBRA 1复合物的形成和增强LC 3-I脂质化形成LC 3-II来重新激活自噬。此外，海藻糖诱导的线粒体自噬和网状吞噬选择性地去除有缺陷的线粒体和应激的ER。目的二是研究海藻糖如何消除p70 S6 K1对自噬的抑制作用，以及p70 S6 K1介导的糖尿病胚胎病的机制。我们的工作假设是，海藻糖通过破坏p70 S6 K1和Beclin-1之间的关联来消除p70 S6 K1对自噬起始复合物的抑制，并且蛋白激酶C α（PKC α）激活p70 S6 K1，其负责糖尿病胚胎病中受损的自噬和NTD形成。目的3探讨AMBRA 1表达的调控机制及其在糖尿病胚胎病自噬和NTD预防中的作用。我们将检验海藻糖通过增加其mRNA稳定性恢复AMBRA 1表达的假设，以及恢复AMBRA 1表达足以重新激活自噬，从而防止糖尿病妊娠中NTD的形成。

DESCRIPTION (provided by applicant): Congenital malformations occur in up to 10% of babies born to diabetic women. Optimal glycemic control is difficult to achieve and maintain, and even transient exposure to hyperglycemia can cause malformations. This project is formulated on the basis of our strong preliminary data. We have found 1) maternal diabetes impairs autophagy and increases the accumulation of defective mitochondria, dysfunctional proteins and swollen endoplasmic reticulum (ER) in the developing neuroepithelium; 2) the non-toxic autophagy activator, trehalose, reverses diabetes-induced autophagy impairment and neural tube defects (NTDs); 3) Fluorescein isothiocyanate (FITC)-labeled trehalose binds to autophagy promoting factors, Beclin-1 and ATG12, and induces selective autophagy; 4) PKCa gene deletion, a p70S6K1 inhibitor and overexpression of an autophagy promoting factor, AMBRA1, in the neural tube, all reduce diabetes-induced NTDs. We test a novel hypothesis that trehalose activates autophagy by re- assembling diabetes-disrupted autophagy initiating complexes, removing the p70S6K1's inhibition and restoring AMBRA1 expression. Both deletion of the S6K1 gene and overexpression of the AMBRA1 gene in the neuroepithelium re-activate autophagy and restore cellular homeostasis leading to NTD prevention. Aim 1 will determine the mechanisms underlying trehalose induction of selective autophagy and restoration of cellular homeostasis leading to prevention of diabetic embryopathy. We hypothesize that trehalose re-activates autophagy by facilitating the formation of the PI3KC3-Beclin-1-AMBRA1 complex and enhancing LC3-I lipidation to form LC3-II. Furthermore, trehalose-induced mitophagy and reticulophagy selectively remove defective mitochondria and stressed ER. Aim 2 will determine how trehalose removes p70S6K1's inhibition on autophagy and the mechanism underlying p70S6K1-mediated diabetic embryopathy. Our working hypothesis is that trehalose removes p70S6K1's inhibition on autophagy initiating complexes by disrupting the association between p70S6K1 and Beclin-1, and that protein kinase C alpha (PKCa) activates p70S6K1, which is responsible for impaired autophagy and NTD formation in diabetic embryopathy. Aim 3 will determine the regulatory mechanism of AMBRA1 expression and its role in autophagy and NTD prevention in diabetic embryopathy. We will test the hypothesis that trehalose restores AMBRA1 expression by increasing its mRNA stability, and that restoring AMBRA1 expression is sufficient to re-activate autophagy, which prevents NTD formation in diabetic pregnancies.