Molecular Dissection of Reactive Astrogliosis: STAT3

描述（由申请方提供）：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）都会触发星形胶质细胞的变化，称为反应性星形胶质细胞增生。反应性星形胶质细胞的作用还没有很好地确定。体外刺激的星形胶质细胞可以产生多种分子，包括促炎和抗炎调节剂，以及细胞毒性和神经保护分子。因此，有害和有益的影响都归因于反应性星形胶质细胞。我们的中心假设是，在对CNS损伤的反应过程中，反应性星形胶质细胞可以以上下文依赖的方式发挥可能对临床结果有益或有害的作用，并受特定的细胞间和细胞内信号传导机制的调节。调节活性星形胶质细胞在体内响应特定情况下的活动的信号传导机制还不清楚。我们以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，并表明这些细胞在体内脑或脊髓损伤后在限制炎症和保护组织方面发挥关键作用。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，我们已经开发了条件性基因缺失或敲除技术（CKO）的星形胶质细胞使用的Cre/loxP系统的调节下的小鼠GFAP启动子在转基因小鼠。在这里，我们建议确定选择性删除STAT 3的影响，STAT 3是一种细胞内信号转导子，被认为是反应性星形胶质细胞增生的调节因子。我们将研究脊髓损伤（SCI）和体外制剂使用相结合的定量形态学和生化分析。我们的初步数据显示，星形胶质细胞STAT 3-CKO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。SCI后，星形胶质细胞STAT 3- CKO小鼠的反应性星形胶质细胞增生减弱，瘢痕形成被破坏。本研究基于我们的初步发现，通过研究三个特定的目的来确定星形胶质细胞STAT 3-CKO的作用：（1）在体内对星形胶质细胞反应性和SCI后瘢痕形成的定量测量以及在体外对各种调节信号通路的影响;（2）对炎症反应、损伤大小及脊髓损伤后短期运动行为的影响;以及影响炎症和细胞毒性的分子的体外星形胶质细胞表达;（3）对脊髓损伤后轴突再生、炎症反应和长期运动行为的影响，以及在体内和体外产生抑制轴突再生和炎性细胞迁移的分子。这一发现将提供有关SCI后调节星形胶质细胞增生的信号机制的基本信息。这些机制信息对于理解决定SCI后功能结果的细胞和分子相互作用是必不可少的，并将有助于确定保证潜在治疗操作靶向的关键途径和分子。公共卫生相关性：脊髓损伤具有毁灭性的后果，很少或没有治疗选择。反应性星形胶质细胞的瘢痕形成是脊髓损伤的一个突出特征，有害和有益的作用都归因于反应性星形胶质细胞。本文提出的工作将通过确定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传导机制来有益于公共卫生，并且可以有针对性地进行治疗操作以改善结果。

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.