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退化和阻碍轴突再生的机制。我们的具体目标是建立新的和创新的模型来研究反应性星形胶质细胞表达的特定分子所扮演的角色，反应性星形胶质细胞是中枢神经系统(CNS)局部组织对损伤和疾病反应的主要细胞类型。这些模型将有助于确定决定反应性星形胶质细胞的生物学和功能的关键分子。 已经描述了许多由反应性星形胶质细胞产生的效应分子，但这些细胞所扮演的角色还没有很好地确定。关于反应性星形胶质细胞的不同功能是如何通过特定的分子触发和执行的，机制信息很少。我们之前在转基因小鼠中建立了一个消融反应性星形胶质细胞的模型，并表明这些细胞对于中枢神经系统损伤后的局部神经保护、血脑屏障修复和炎症调节是必不可少的。我们的下一个目标是确定这些表型的分子机制。在这里，我们建议建立转基因模型，用于靶向和可调控地消融反应性星形胶质细胞表达的特定分子分子。这些模型使用Cre/loxP系统，其中细菌Cre重组酶I切除插入失活目标基因的loxP位点之间的DNA序列。 目的建立并鉴定表达胶质纤维蛋白(GFAP)启动子Cre的转基因小鼠，从而将Cre靶向反应性星形胶质细胞。目标2是通过使用四环素可调节启动子系统获得额外水平调控的转基因小鼠。在这些小鼠中，Cre在星形胶质细胞中的表达在发育过程中会受到抑制，而在成年小鼠中会被激活。转基因品系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其使用。目标3是通过去除表达星形胶质细胞的GFAP中的一个关键调控分子来证明原理。STAT3是JAK-STAT家族的细胞内信号转导分子，参与介导MAY细胞对多种细胞因子和生长因子的增殖和分化反应。STAT3信号与星形胶质细胞发育和反应性星形胶质细胞增多症有关。在STAT3基因中插入loxP位点的小鼠是可用的，并将与来自AIMS 1和2的Cre小鼠杂交。STAT3缺陷的星形胶质细胞将在体外和体内实验性中枢神经系统损伤后评估其反应能力。突变小鼠中枢神经系统损伤后预测的星形胶质细胞损伤失败的后果将在体内进行研究。 AIMS 1和AIMS 2的模型将允许测试关于反应性星形胶质细胞表达的特定分子在中枢神经系统损伤后反应性星形胶质细胞的生物学和功能中所扮演的角色的许多假说。这些模型将对我们自己和合作实验室未来的许多不同研究有用。AIM 3将开始这一进程。短期和长期的研究结果将有助于了解决定中枢神经系统损伤后功能结果的细胞和分子机制的基本信息。这类机制信息对于制定合理的治疗策略是必不可少的。

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.