Autophagy and its Regulation in Diabetic Embryopathy

描述(由申请人提供)：糖尿病妇女所生婴儿中有高达10%会发生先天性畸形。最优的血糖控制很难实现和维持，即使是短暂的高血糖暴露也会导致畸形。这个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，并增加发育中的神经上皮中有缺陷的线粒体、功能障碍的蛋白质和肿胀的内质网(ER)的积聚；2)无毒的自噬激活剂海藻糖逆转糖尿病引起的自噬损害和神经管畸形(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ATG12结合，诱导选择性自噬；4)PKCA基因缺失，p70S6K1抑制因子和自噬促进因子AMBRA1在神经管中过表达，可减少糖尿病引起的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体，消除p70S6K1‘S抑制和恢复AMBRA1的表达来激活自噬。S6K1基因的缺失和AMBRA1基因在神经上皮细胞中的过表达都会重新激活自噬，恢复细胞内的平衡，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以预防糖尿病胚胎病变的机制。我们假设海藻糖通过促进PI3KC3-Beclin-1-AMBRA1复合体的形成和促进Lc3-I的脂化形成Lc3-II来重新激活自噬。此外，海藻糖诱导的有丝分裂吞噬和网状吞噬选择性地移除有缺陷的线粒体和应激的内质网。目的2确定海藻糖如何解除p70S6K1‘S对自噬的抑制，以及p70S6K1介导的糖尿病胚胎病变的机制。我们的工作假设是海藻糖通过破坏p70S6K1和Beclin-1之间的联系来解除p70S6K1‘S对自噬启动复合体的抑制，而蛋白激酶Cα(PKCA)激活p70S6K1，这是导致糖尿病胚胎病变自噬和NTD形成障碍的原因。目的3研究AMBRA1基因表达的调控机制及其在糖尿病胚胎病变自噬和NTD预防中的作用。我们将测试这样的假设，即海藻糖通过增加AMBRA1的mRNA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.