Autophagy and its Regulation in Diabetic Embryopathy

描述（由申请人提供）：糖尿病妇女所生婴儿的先天性畸形发生率高达10%。最佳血糖控制是难以实现和维持的，即使短暂的高血糖也会导致畸形。这个项目是在我们强有力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，增加发育中的神经上皮中有缺陷的线粒体、功能失调的蛋白质和肿胀的内质网（ER）的积累；2)无毒自噬激活剂海藻糖可逆转糖尿病诱导的自噬损伤和神经管缺陷（NTDs）；3)异硫氰酸荧光素标记海藻糖与自噬促进因子Beclin-1和ATG12结合，诱导选择性自噬；4) PKCa基因缺失、p70S6K1抑制剂和神经管中自噬促进因子AMBRA1的过表达均可降低糖尿病诱导的NTDs。我们验证了一个新的假设，即海藻糖通过重组糖尿病破坏的自噬启动复合物，去除p70S6K1的抑制并恢复AMBRA1的表达来激活自噬。神经上皮中S6K1基因的缺失和AMBRA1基因的过表达都能重新激活自噬，恢复细胞稳态，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稳态的机制，从而预防糖尿病胚胎病。我们假设海藻糖通过促进PI3KC3-Beclin-1-AMBRA1复合物的形成和促进LC3-I脂化形成LC3-II来重新激活自噬。此外，海藻糖诱导的线粒体吞噬和网状吞噬选择性地去除缺陷线粒体和应激内质网。目的2将确定海藻糖如何去除p70S6K1对自噬的抑制，以及p70S6K1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p70S6K1和Beclin-1之间的联系来消除p70S6K1对自噬启动复合物的抑制，并且蛋白激酶C α (PKCa)激活p70S6K1，这是糖尿病胚胎病中自噬受损和NTD形成的原因。Aim 3将确定AMBRA1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。我们将验证海藻糖通过增加AMBRA1 mRNA的稳定性来恢复AMBRA1的表达，并且恢复AMBRA1的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.