Microtubule Stability in Lung Endothelium

描述（由申请人提供）：肺微血管内皮形成一个限制性屏障，允许适当的气体交换。炎症介质和血管通透性增加化合物通过重组内皮细胞骨架、细胞-细胞和细胞基质相互作用引起细胞边界和内皮间隙的收缩。炎症介质诱导内皮细胞骨架发生两个关键变化，包括皮质肌动蛋白边缘重组为应力纤维，以及微管的分解和重组。尽管它们在控制内皮细胞形状方面很重要，但对控制微管组装和拆卸的细胞内信号知之甚少。微管相关蛋白调节微管动力学。Tau是一种主要的神经元微管相关蛋白，可促进轴突微管的组装、稳定性和捆绑。Tau过度磷酸化降低了Tau微管结合能力并导致神经原纤维缠结形成，这是阿尔茨海默病的病理严重程度标志。蛋白激酶A通过磷酸化Tau蛋白的214、262和356丝氨酸参与神经原纤维缠结的形成。就在最近，非神经元Tau蛋白被发现调节内皮细胞的微管动力学。在内皮细胞中，可溶性腺苷酸环化酶活性与Tau丝氨酸214磷酸化和微管重组有关，初步数据表明Tau丝氨酸214磷酸化可从内皮微管中释放Tau蛋白。有趣的是，细菌进化出了将可溶性腺苷酸环化酶插入内皮细胞并增加通透性的毒性机制。由于多种不同的激酶可以磷酸化Tau蛋白，腺苷酸环化酶活性诱导Tau蛋白过度磷酸化和内皮屏障破坏的机制尚不清楚。同样不清楚细菌腺苷酸环化酶是否通过tau依赖机制诱导内皮细胞高通透性。因此，本应用验证了细菌腺苷酸环化酶毒素诱导Tau丝氨酸-214磷酸化促进微管分解并增加微血管内皮通透性的总体假设。具体目的将验证相关假设：[1]细菌可溶性腺苷酸环化酶毒素激活磷酸化Tau丝氨酸214的PKA；Tau ser-214的磷酸化足以分解微管并增加渗透率。这些研究的完成不仅会影响对内皮通透性和细菌诱导的急性肺损伤的理解，而且在神经退行性疾病领域也具有重要意义。在目前的提议中，我们将严格测试细菌腺苷酸环化酶是否产生cAMP信号，导致Tau ser-214磷酸化，足以解聚微管并增加内皮细胞的通透性，并且由于PKA磷酸化是阿尔茨海默病中已知的病理生理事件，我们的研究将解决PKA激活导致Tau过度磷酸化的假定机制。

DESCRIPTION (provided by applicant): Pulmonary microvascular endothelium forms a restrictive barrier to allow proper gas exchange. Inflammatory mediators and vascular permeability-increasing compounds cause retraction of cell borders and inter-endothelial gaps by reorganizing the endothelial cytoskeleton, cell-cell, and cell matrix interactions. Inflammatory mediators induce two critical changes in the endothelial cell cytoskeleton, including reorganization of the cortical actin rim into stress fibers, and disassembly and reorganization of microtubules. Despite their importance in control of endothelial cell shape, relatively little is known about intracellular signals that control microtubule assembly and disassembly. Microtubule-associated proteins regulate microtubule dynamics. Tau is a major neuronal microtubule-associated protein that promotes assembly, stability, and bundling of axonal microtubules. Tau hyper-phosphorylation reduces Tau microtubule binding-ability and causes neurofibrillary tangle formation, a pathologic severity marker of Alzheimer's disease. Protein Kinase A contributes to neurofibrillary tangle formation by phosphorylating Tau at serines 214, 262, and 356. Just recently, non-neuronal Tau has been found to regulate microtubule dynamics in endothelium. In endothelial cells, soluble adenylyl cyclase activity has been associated with Tau serine 214 phosphorylation and microtubule reorganization, and preliminary data suggest that Tau serine 214 phosphorylation releases Tau from endothelial microtubules. Interestingly, bacteria have evolved toxicity mechanisms that insert soluble adenylyl cyclases into endothelial cells and increase permeability. Since multiple different kinases can phosphorylate Tau, the mechanism by which adenylyl cyclase activity induces Tau hyperphosphorylation and endothelial barrier disruption is unknown. It is similarly unclear whether bacterial adenylyl cyclases induce endothelial hyperpermeability by a Tau-dependent mechanism. Therefore, the present application tests the overall hypothesis that bacterial adenylyl cyclase toxins induce Tau serine-214 phosphorylation that promotes microtubule disassembly and increases microvascular endothelial permeability. Specific aims will test the related hypotheses that: [1] Bacterial soluble adenylyl cyclase toxins activate PKA that phosphorylates Tau serine 214; and [2] Phosphorylation of Tau ser-214 is sufficient to disassemble microtubules and increase permeability. Completion of these studies will not only impact the understanding of endothelial permeability and bacterial-induced acute lung injury, but will also be significant in the field of neurodegenerative diseases. In the present proposal, we will rigorously test whether bacterial adenylyl cyclases generate a cAMP signal that results in phosphorylation of Tau ser-214 sufficient to depolymerize microtubules and increase endothelial permeability, and as PKA phosphorylation of Tau is a known pathophysiological event in Alzheimer's disease, our studies will resolve a putative mechanism responsible for PKA activation that results in Tau hyperphosphorylation. PUBLIC HEALTH RELEVANCE: Studies proposed in this application will address whether the microtubule-associated protein Tau plays an important role in maintaining lung vascular barrier integrity. This is very relevant to public health since these studies could help design new pharmacologic strategies to treat pathologies in which the lung vascular barrier is damaged, like acute respiratory distress syndrome and sepsis.