Astrocytic TGFB Signaling Pathways and Neuroinflammation Following Stroke

描述（由申请人提供）：中风是世界上导致死亡和残疾的主要原因之一。FDA唯一批准的治疗中风的药物是组织纤溶酶原激活剂，它只在中风后大约4.5小时内有效。一种具有较长治疗窗口期的有希望的策略是限制中风后过度的神经炎症。过度的炎症使梗死面积扩大，并使炎症反应长期存在。在允许其有益作用发生后促进神经炎症的解决可能改善卒中的预后。转化生长因子β (TGF？'s)是主要的调节细胞因子，以非活性形式分泌并隔离在细胞外基质中。它们主要具有抗炎作用，中风后几天它们的信号会升高。星形胶质细胞是对TGF？在中风后的亚急性期。因为星形胶质细胞是与神经元、血管和免疫细胞相互作用的神经胶质细胞，它们很好地协调了神经炎症反应。然而TGF吗?在星形胶质细胞中的调控和功能尚不完全清楚。我们的初步数据表明，脑卒中边界星形胶质细胞产生的血栓反应蛋白-1激活TGF？在细胞外基质中，这种增加上调了TGF？以及星形胶质细胞中的血栓反应蛋白1。此外，抑制星形胶质细胞TGF？信号通路使炎症反应增加约一倍，降低梗死附近存活神经元中具有神经保护作用的Akt信号通路，并使卒中后的预后恶化。因此，我们的中心假设是星形胶质细胞利用TGF？在中风后亚急性期触发一个正反馈循环，导致活性TGF？，从而减少神经炎症，改善治疗效果。我们的目的是确定星形细胞TGF？信号传导是触发一个正反馈循环的必要和充分条件，从而增加活性TGF？中风后亚急性发作。我们还旨在确定是否增加整体或星形细胞TGF？超过基线的信号足以减少神经炎症和改善中风后的功能结局。确定星形细胞TGF？信号是这个正反馈回路所必需的，我们将使用双转基因模型，减少TGF？并通过激活全局TGF?来补充这一缺陷。我们还将确定这个星形细胞TGF？通过不同的双转基因模型，上调星形细胞TGF？信号。我们将使用ELISA、Western blot、免疫组织化学和qPCR检测TGF？和血小板反应蛋白-1，以及TGF？在这些模型中。此外，我们将利用这些模型来确定TGF？信号提供减少神经炎症和改善中风后的功能结局。无论我们的假设是否正确，所提出的实验将更好地理解星形胶质细胞用于影响中风后神经炎症的机制，这也适用于其他类型的中枢神经系统炎症。

DESCRIPTION (provided by applicant): Stroke is one of the leading causes of death and disability in the world. The only FDA approved therapy for stroke is tissue plasminogen activator and it is only effective within approximately 4.5 hours following stroke. One promising strategy with a longer treatment window is limiting excessive neuroinflammation following stroke. Excessive inflammation worsens infarct sizes and perpetuates the inflammatory response. Promoting resolution of neuroinflammation after allowing its beneficial effects to occur may improve outcome in stroke. Transforming growth factor betas (TGF?'s) are master regulatory cytokines that are secreted in an inactive form and sequestered in the extracelluar matrix. They are primarily anti-inflammatory and their signaling is elevated days after stroke. Astrocytes are one of the cell types responding to increased levels of TGF? during this subacute period after stroke. Because astrocytes are glial cells that interact with neurons, blood vessels, and immune cells, they are well situated to coordinate neuroinflammatory responses. However TGF?'s regulation and function in astrocytes is not completely understood. Our preliminary data suggests that thrombospondin-1 production by astrocytes in the stroke border activates TGF? in the extracellular matrix, and the increase upregulates TGF? and thrombospondin-1 in astrocytes. Additionally, inhibiting astrocytic TGF? signaling approximately doubles the inflammatory response, decreases neuroprotective Akt signaling in surviving neurons adjacent to the infarct, and worsens outcomes after stroke. Our central hypothesis is therefore that astrocytes utilize TGF? in the subacute time period after stroke to trigger a positive feedback loop that leads to higher levels of active TGF?, which then reduces neuroinflammation and improves outcomes. We aim to determine whether astrocytic TGF? signaling is necessary and sufficient to trigger a positive feedback loop that increases active TGF? subacutely after stroke. We also aim to determine whether increasing global or astrocytic TGF? signaling over baseline is sufficient to decrease neuroinflammation and improve functional outcomes after stroke. To determine whether astrocytic TGF? signaling is necessary for this positive feedback loop we will use a double transgenic model with reduced TGF? specifically in astrocytes and supplement this deficit by activating global TGF?. We will also determine whether this astrocytic TGF? signaling is sufficient for this positive feedback loop with a different double transgenic model, upregulating astrocytic TGF? signaling. We will use ELISA, Western blot, immunohistochemistry, and qPCR to measure global levels of TGF? and thrombospondin-1, and signaling pathways of TGF? in each of these models. In addition, we will use these models to determine which conditions of TGF? signaling provide reduced neuroinflammation and improved functional outcomes following stroke. Regardless of our hypothesis being correct, the proposed experiments will provide a better understanding of the mechanisms that astrocytes use to influence neuroinflammation following stroke, which be applicable to other types of central nervous system inflammation as well.