TGFbeta signaling, reactive astrogliosis and function after stroke

描述(申请人提供)：中风是美国长期残疾的主要原因。尽管急性血运重建疗法可以用于流产或减轻中风负担，但目前还没有药物可以改善中风发生后的恢复。炎症反应是此类治疗的一个有希望的靶点，因为它发生在中风后的几天和几周，可能是有害的，也可能是有益的。一个主要的悬而未决的问题是，受损的大脑是如何调节免疫反应的，以及是否有分子途径可以通过调节整体免疫反应来对功能恢复施加有益或有害的影响。星形胶质细胞是大脑损伤反应的关键组成部分--所谓的“反应性星形胶质细胞”在脑损伤后随处可见。它们也越来越被认为是大脑固有免疫系统的关键组成部分。我们建议询问星形胶质细胞中的转化生长因子β信号是否调节中风后的炎症，因为它是免疫反应的主要调节因子。转化生长因子可消除损伤后的免疫反应，并促使免疫细胞表型向炎症较少的状态发展。我们的初步实验表明，中风后大脑中的转化生长因子信号增加，持续数周，并发生在反应性星形胶质细胞中。为了测试转化生长因子S在反应性星形胶质细胞中的功能是否反映了它在其他类型的免疫细胞中的作用，我们构建了转化生长因子β信号仅在星形胶质细胞中减少的小鼠。我们发现，这些小鼠的原代星形胶质细胞在缺氧-葡萄糖剥夺后表现出更多的“促炎”表型，而小鼠本身在中风后表现出更高的炎症反应。基于这一数据，我们假设卒中后，转化生长因子信号(1)发生在反应性星形胶质细胞中，(2)限制炎症反应，(3)促进功能恢复。我们计划在三个具体目标上检验我们的假设。在目标1中，我们将使用报告小鼠和免疫组织化学来确定中风后转化生长因子信号的模式。我们假设卒中后数周内对转化生长因子-β的反应增强，而反应性星形胶质细胞在卒中后对转化生长因子-β有反应。在目标2中，我们将利用遗传学和药理学方法，通过体内和体外实验来靶向星形胶质细胞中的转化生长因子信号，以测试星形胶质细胞转化生长因子β信号在缺血神经炎性反应中的作用。我们假设星形胶质细胞的转化生长因子信号驱动中风免疫反应的解决。在目标3中，我们将使用一个遗传小鼠模型来研究中风诱导的星形胶质细胞转化生长因子信号对功能恢复是有利还是不利。我们预测星形胶质细胞的转化生长因子信号可促进卒中的康复。随着拟议实验的完成，我们将确定中风后转化生长因子反应的长度和细胞特异性。我们将深入了解星形胶质细胞如何影响中风的免疫反应，以及反应性星形胶质细胞的功能多样性。我们的发现可能会导致针对大脑免疫反应的治疗，并使中风后几天内就医的患者受益。

DESCRIPTION (provided by applicant): Stroke is the leading cause of long-term disability in the United States. Although acute revascularization therapies can be used to abort or reduce stroke burden, there are currently no drugs that improve recovery after a stroke has happened. The inflammatory response is a promising target for such therapies as it occurs in the days and weeks after a stroke and can be both detrimental and beneficial. A major unanswered question is how the injured brain modulates immune responses, and if there are molecular pathways that can be utilized to exert beneficial or limit detrimental effects on functional recovery via modulating the overall immune response. Astrocytes are a key component of the brain's injury response - so-called "reactive astrocytes" are ubiquitous after brain injury. They are also increasingly recognized as key components of the brain's innate immune system. We propose to ask if Transforming Growth Factor Beta (TGF¿) signaling in astrocytes modulates inflammation after stroke because it is a master regulator of immune responses. TGF¿ can resolve immune responses after injury and drive immune cell phenotypes towards less inflammatory states. Our preliminary experiments show that TGF¿ signaling is increased in the brain after stroke, persists for weeks, and occurs in reactive astrocytes. To test if TGF¿'s function in reactive astrocytes mirror its role in other types of immune cells we constructed mice in which TGF¿ signaling is decreased only in astrocytes. We have found that primary astrocytes from these mice exhibit a more "pro-inflammatory" phenotype after oxygen- glucose deprivation, and the mice themselves demonstrate increased inflammatory responses after stroke. Based on this data we hypothesize that after stroke, TGF¿ signaling (1) occurs in reactive astrocytes, (2) limits the inflammatory response, and (3) improves functional recovery. We plan to test our hypothesis in three Specific Aims. In Aim 1 we will use reporter mice and immunohistochemistry to determine patterns of TGF¿ signaling after stroke. We hypothesize that there are increased responses to TGF-¿ for weeks after stroke, and that reactive astrocytes are responding to TGF¿ after stroke. In Aim 2 we will test the function of astrocytic TGF¿ signaling in the neuroinflammatory response to ischemia, using genetic and pharmacological approaches and in vivo and in vitro experiments to target TGF¿ signaling in astrocytes. We hypothesize that astrocytic TGF¿ signaling drives resolution of the immune response to stroke. In Aim 3 we will use a genetic mouse model to ask if stroke-induced astrocytic TGF¿ signaling is beneficial or detrimental for functional recovery. We predict that astrocytic TGF¿ signaling improves recovery from stroke. With the completion of the proposed experiments we will have defined the length and cell specificity of TGF¿ responses after stroke. We will gain insight into how astrocytes influence the immune response to stroke, and into the functional diversity of reactive astrocytes. Our findings may lead to therapies that will target the brain's immune responses and benefit patients who present for medical care in the days after stroke.