Molecular Dissection of Reactive Astrogliosis: STAT3

描述(申请人提供)：所有中枢神经系统(CNS)损伤，包括创伤、感染、缺血和退行性疾病，都会引发星形胶质细胞的变化，称为反应性星形胶质细胞增生症。反应性星形胶质细胞的作用尚未完全确定。在体外刺激的星形胶质细胞可以产生各种各样的分子，包括促炎和抗炎调节因子，以及细胞毒性和神经保护分子。因此，反应性星形胶质细胞既有有害的影响，也有有益的影响。我们的中心假设是，在对中枢神经系统侮辱的反应中，反应性星形胶质细胞可以以一种依赖于上下文的方式发挥对临床结果有利或有害的影响，并受特定的细胞间和细胞内信号机制调节。调节反应性星形胶质细胞在体内特定情况下活动的信号机制还不是很清楚。我们之前的工作使用转基因小鼠模型来消融反应性星形胶质细胞，并表明这些细胞在脑或脊髓损伤后的体内抑制炎症和保护组织方面发挥着关键作用。我们的下一个目标是确定调节反应性星形胶质细胞特定活动的分子机制。为此，我们开发了在转基因小鼠中使用Cre/loxP系统在小鼠GFAP启动子的调控下对星形胶质细胞进行条件性基因缺失或敲除技术(CKO)。在这里，我们建议确定选择性删除STAT3的效果，STAT3是一种细胞内信号转导，被认为是反应性星形胶质细胞增生的调节因子。我们将结合定量形态和生化分析来研究脊髓损伤(SCI)和体外准备。我们的初步数据显示，患有星形胶质细胞STAT3-CKO的小鼠具有正常大小和细胞学的CNS，并且星形胶质细胞以正常数量生成。脊髓损伤后，星形胶质细胞STAT3-CKO小鼠的反应性星形胶质细胞增生减弱，瘢痕形成被破坏。这一建议基于我们的初步发现，通过研究三个特定目标来确定星形胶质细胞STAT3-CKO的作用：(1)定量测量体内脊髓损伤后星形胶质细胞的反应性和瘢痕形成，以及体外各种调控信号通路；(2)体内脊髓损伤后炎症、病变大小和短期运动行为，以及星形胶质细胞体外影响炎症和细胞毒性的分子的表达；(3)体内脊髓损伤后轴突再生、炎症和长期运动行为的影响，以及体内和体外抑制轴突再生和炎性细胞迁移的分子的产生。这些发现将为调控脊髓损伤后星形胶质细胞增生的信号机制提供基本信息。这些机制信息对于理解决定脊髓损伤后功能结果的细胞和分子相互作用是必不可少的，并将有助于识别关键的通路和分子，为潜在的治疗操作提供靶向。与公共卫生相关：脊髓损伤具有毁灭性的后果，几乎没有治疗选择。反应性星形胶质细胞形成瘢痕是脊髓损伤的一个显著特征，反应性星形胶质细胞既有有害的影响，也有有益的影响。本文提出的这项工作将通过识别调节脊髓损伤后反应性星形胶质细胞特定功能的分子信号机制来造福公众健康，并可作为治疗操作的靶向，以改善结果。

DESCRIPTION (provided by applicant): All central nervous system (CNS) insults including trauma, infection, ischemia and degenerative disease trigger changes in astroglia known as reactive astrogliosis. The roles of reactive astroglia are not well established. Astroglia stimulated in vitro can produce a wide variety of molecules including both pro- and anti-inflammatory regulators, as well as cytotoxic and neuroprotective molecules. Accordingly, both harmful and beneficial effects have been attributed to reactive astrocytes. Our central hypothesis is that during the response to CNS insults, reactive astrocytes can exert effects that may be either beneficial or detrimental to clinical outcome in a manner that is context dependent and is regulated by specific inter- and intra-cellular signaling mechanisms. The signaling mechanisms that regulate activities implemented by reactive astrocytes in response to specific situations in vivo are not well understood. Our previous work used a transgenic mouse model to ablate reactive astrocytes and showed that these cells play pivotal roles in restricting inflammation and protecting tissue after brain or spinal cord injury in vivo. Our next goal is to identify molecular mechanisms that regulate specific activities of reactive astrocytes. To do so we have developed conditional gene deletion or knockout technology (CKO) for astrocytes using the Cre/loxP system under regulation of the mouse GFAP promoter in transgenic mice. Here we propose to determine the effects of selectively deleting STAT3, an intracellular signal transducer that has been implicated as a regulator of reactive astrogliosis. We will study spinal cord injury (SCI) and in vitro preparations using a combination of quantitative morphological and biochemical analyses. Our preliminary data show that mice with astroglial STAT3-CKO have CNS of normal size and cytology, and that astrocytes are generated in normal numbers. After SCI, reactive astrogliosis is attenuated and scar formation is disrupted in mice with astroglial STAT3- CKO. This proposal builds on our preliminary findings by investigating three specific aims that will determine the effects of astroglial STAT3-CKO: (1) on quantitative measures of astrocyte reactivity and scar formation after SCI in vivo and on various regulatory signaling pathways in vitro; (2) on inflammation, lesion size and short-term motor behavior after SCI in vivo, and on astrocyte expression in vitro of molecules that influence inflammation and cytotoxicity; and (3) on axon regeneration, inflammation and long-term motor behavior after SCI in vivo, and on the production in vivo and in vitro of molecules that inhibit both axon regeneration and inflammatory cell migration. The findings will provide fundamental information about signaling mechanisms that regulate astrogliosis after SCI. Such mechanistic information is essential for understanding the cellular and molecular interactions that determine functional outcome after SCI, and will help to identify key pathways and molecules that warrant targeting for potential therapeutic manipulation. PUBLIC HEALTH RELEVANCE: Spinal cord injury has devastating consequences and little or no treatment options. Scar formation by reactive astrocytes is a prominent feature of spinal cord injury, and both harmful and beneficial effects have been attributed to reactive astrocytes. The work proposed here will benefit public health by identifying molecular signaling mechanisms that regulate specific functions of reactive astrocytes after spinal cord injury and can be targeted for therapeutic manipulation to improve outcome.