Molecular Dissection of Reactive Astrocyte Biology

描述（由申请人提供）： 我们的长期目标是了解和对抗脑和脊髓损伤后导致细胞变性和阻碍轴突再生的机制。我们的具体目标是产生新的和创新的模型，以研究由反应性星形胶质细胞表达的特定分子所发挥的作用，星形胶质细胞是中枢神经系统（CNS）损伤和疾病的局部组织反应中的主要细胞类型。这些模型将有助于确定决定反应性星形胶质细胞生物学和功能的关键分子。 已经描述了由反应性星形胶质细胞产生的许多效应分子，但是这些细胞所起的作用还没有很好地定义。关于反应性星形胶质细胞的不同功能如何通过特定分子触发和实现的机制信息很少。我们先前开发了一种用于消融转基因小鼠中反应性星形胶质细胞的模型，并表明这些细胞对于CNS损伤后的局部神经保护、血屏障修复和炎症调节至关重要。我们的下一个目标是确定这些表型的分子机制。在这里，我们建议开发转基因模型的反应性星形胶质细胞表达的特定分子的靶向和可调控的消融。这些模型使用Cre/loxP系统，其中细菌Cre重组酶I切除插入到靶向灭活的基因中的loxP位点之间的DNA序列。 目的I是产生和表征转基因小鼠表达Cre从胶质细胞胶质细胞蛋白（GFAP）启动子，从而将Cre反应性星形胶质细胞。目的2是产生转基因小鼠，其中通过使用四环素可调控启动子系统实现额外水平的调控。在这些小鼠中，星形胶质细胞中的Cre表达将在发育过程中被抑制，并在成年小鼠中被激活。将通过在单细胞水平上证明Cre活性靶向反应性星形胶质细胞的特异性来验证转基因系的使用。目的3是通过从表达GFAP的星形胶质细胞特异性地消融关键调节分子来证明原理的证据。STAT 3是JAK-STAT家族的细胞内信号转导子，参与介导多种细胞类型中对各种细胞因子和生长因子的增殖和分化应答。STAT 3信号转导与星形胶质细胞发育和反应性星形胶质细胞增多症有关。可获得在STAT 3基因中插入loxP位点的小鼠，并将其与目的1和2的Cre小鼠杂交。将在体外和在体内实验性CNS损伤后评价STAT 3缺陷型星形胶质细胞的反应能力。将在体内研究突变小鼠CNS损伤后预测的星形胶质细胞增多失败的后果。 目标1和2的模型将允许测试许多关于反应性星形胶质细胞表达的特定分子在CNS损伤后反应性星形胶质细胞的生物学和功能中所起作用的假设。这些模型将有助于我们自己和合作实验室的许多不同的未来研究。目标3将开始这一进程。从短期和长期来看，这些发现将有助于了解决定中枢神经系统损伤后功能结局的细胞和分子机制的基本信息。这类机制信息对于基于理性的治疗策略的发展是必不可少的。

DESCRIPTION (provided by applicant): Our long-term objective is to understand and counteract mechanisms that lead to cellular degeneration and impede axon regeneration after brain and spinal cord injury. Our specific aims here are to generate new and innovative models to study the roles played by specific molecules that are expressed by reactive astrocytes, a principal cell type in the local tissue response to injury and disease in the central nervous system (CNS). These models will help to identify key molecules that determine the biology and function of reactive astrocytes. Numerous effector molecules produced by reactive astrocytes have been described, but the roles played by these cells are not well defined. Little mechanistic information is available about how the different functions of reactive astrocytes are triggered and implemented through specific molecules. We previously developed a model for the ablation of reactive astrocytes in transgenic mice and showed that these cells are essential for local neuroprotection, blood brat barrier repair and regulation of inflammation after CNS injury. Our next goal is to identify molecular mechanisms for these phenotypes. Here, we propose to develop transgenic models for the targeted and regulatable ablation of specific molecules molecules expressed by reactive astrocytes. These models use the Cre/loxP system, where bacterial Cre recombinase I excises DNA sequences between loxP sites inserted into genes targeted for inactivation. Aim I is to generate and characterize transgenic mice that express Cre from the glial fibrillary protein (GFAP) promoter, thus targeting Cre to reactive astrocytes. Aim 2 is to generate transgenic mice in which an additional level of regulation is achieved by using a tetracycline regulatable promoter system. In these mice, Cre expression in astrocytes will be suppressed during development and activated in adult mice. Transgenic lines will be validated for use by demonstrating the specificity of the targeting of Cre activity to reactive astrocytes at the single cell level. Aim 3 is to demonstrate proof of principle by ablating a crucial regulatory molecule specifically from GFAP expressing astrocytes. STAT3 is an intracellular signal transducer of the JAK-STAT family involved in mediating the proliferation and differentiation responses to various cytokines and growth factors in may cell types. STAT3 signaling has been implicated in both astrocyte development and reactive astrocytosis. Mice with loxP sites inserted into the STAT3 gene are available and will be crossbred with Cre mice from Aims 1 and 2. STAT3-deficient astrocytes will be evaluated in vitro and after experimental CNS injury in vivo for their ability to become reactive. The consequences of the predicted failure of astrocytosis after CNS injury in mutant mice will be studied in vivo. The models from Aims 1 and 2 will allow the testing of many hypotheses about the roles that specific molecules expressed by reactive astrocytes play in the biology and function of reactive astrocytes after CNS injury. These models will be useful for many different future studies in our own and collaborating laboratories. Aim 3 will begin this process. The findings obtained in both the short and long term will contribute fundamental information about cellular and molecular mechanisms that determine functional outcome after CNS injury. Mechanistic information of this kind is essential for the development of rationally based therapeutic strategies.