Molecular Dissection of Reactive Astrogliosis: STAT3

描述（由申请人提供）：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。反应性星形胶质细胞的作用尚未完全确定。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，反应性星形胶质细胞产生了有害和有益的影响。我们的中心假设是，在对中枢神经系统损伤的反应过程中，反应性星形胶质细胞可以以一种依赖于环境的方式对临床结果产生有益或有害的影响，并受特定的细胞间和细胞内信号传导机制的调节。反应性星形胶质细胞在体内特定情况下调控活动的信号机制尚不清楚。我们之前的工作使用转基因小鼠模型切除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限制炎症和保护组织中发挥关键作用。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，我们在转基因小鼠的GFAP启动子调控下，利用Cre/loxP系统开发了星形胶质细胞的条件基因删除或敲除技术（CKO）。在这里，我们建议确定选择性删除STAT3的影响，STAT3是一种细胞内信号换能器，与反应性星形胶质细胞形成的调节有关。我们将使用定量形态学和生化分析相结合的方法研究脊髓损伤（SCI）及其体外制剂。我们的初步数据表明，星形胶质细胞STAT3-CKO小鼠具有正常大小和细胞学的中枢神经系统，星形胶质细胞的数量正常。SCI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。本研究基于我们的初步研究结果，研究了星形胶质细胞STAT3-CKO的三个具体目标：(1)在体内对SCI后星形胶质细胞反应性和疤痕形成的定量测量，以及在体外对各种调节信号通路的影响；(2)体内损伤后的炎症、病变大小和短期运动行为，以及影响炎症和细胞毒性的分子在体外星形胶质细胞的表达；(3)体内脊髓损伤后的轴突再生、炎症和长期运动行为，以及体内和体外抑制轴突再生和炎症细胞迁移的分子的产生。这一发现将为脊髓损伤后星形胶质细胞形成的信号机制提供基础信息。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至关重要，并将有助于确定关键途径和分子，以保证潜在的治疗操作。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形成疤痕是脊髓损伤的一个显著特征，其有害和有益的影响都归因于反应性星形胶质细胞。这里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机制，从而有益于公众健康，并可以针对治疗操作来改善结果。

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.