Astrocytic TGFB Signaling Pathways and Neuroinflammation Following Stroke

描述（由申请人提供）：中风是世界上死亡和残疾的主要原因之一。FDA唯一批准的中风疗法是组织纤溶酶原激活剂，并且它仅在中风后约4.5小时内有效。一种具有较长治疗窗的有希望的策略是限制中风后的过度神经炎症。过度的炎症会扩大梗死面积，并使炎症反应持续存在。在允许其有益作用发生后促进神经炎症的消退可能会改善卒中的结局。转化生长因子β（TGF？是以无活性形式分泌并隔离在细胞外基质中的主要调节细胞因子。它们主要是抗炎的，并且它们的信号在中风后几天升高。星形胶质细胞是一种细胞类型的反应增加的TGF？在中风后的亚急性期由于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，因此它们很好地协调神经炎症反应。然而，TGF？在星形胶质细胞中的调节和功能还不完全清楚。我们的初步数据表明，血小板反应蛋白-1的生产星形胶质细胞在中风的边界激活TGF？在细胞外基质，和增加上调TGF？和血小板反应蛋白-1。此外，抑制星形胶质细胞TGF？信号转导使炎症反应大约加倍，减少了梗死附近存活神经元中的神经保护性Akt信号转导，并降低了中风后的结果。因此，我们的中心假设是星形胶质细胞利用TGF？在中风后的亚急性时期触发正反馈循环，导致更高水平的活性TGF？，从而减少神经炎症并改善结果。我们的目的是确定星形胶质细胞TGF？信号是必要的，足以触发一个积极的反馈回路，增加积极的TGF？中风后的亚急性期我们还旨在确定是否增加全球或星形胶质细胞TGF？超过基线的信号足以减少神经炎症并改善中风后的功能结果。以确定是否星形胶质细胞TGF？信号是必要的，这个正反馈回路，我们将使用一个双转基因模型与减少TGF？特别是在星形胶质细胞中，并通过激活整体TGF β来补充这种缺陷。我们还将确定是否这个星形胶质细胞TGF？信号是足够的，这个正反馈回路与不同的双转基因模型，上调星形胶质细胞TGF？发信号。我们将使用ELISA，蛋白质印迹，免疫组化和qPCR来测量全球TGF？和血小板反应蛋白-1，和信号通路的TGF？in each每of these models模型.此外，我们将使用这些模型，以确定哪些条件下的TGF？信号传导在中风后提供减少的神经炎症和改善的功能结果。无论我们的假设是否正确，所提出的实验将提供对星形胶质细胞用于影响中风后神经炎症的机制的更好理解，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.