Role of Palladin in Regulating Astrocytes

描述(申请人提供)：星形胶质细胞对中枢神经系统损伤的反应是形态上的剧烈变化，导致形成致密的胶质瘢痕，从而抑制轴突再生。“反应性星形胶质细胞”表型的分子基础尚不清楚。我们最近发现了一种新的细胞骨架相关蛋白Palladin，它在维持多种细胞类型的肌动蛋白细胞骨架方面起着至关重要的作用，本研究探讨了Palladin在体外和体内星形胶质细胞对机械损伤的反应中的作用。我们的假设是，Palladin作为一个分子支架来组织肌动蛋白细胞骨架，并促进星形胶质细胞对特定刺激的反应改变细胞形状。我们获得的初步证据表明，在培养的星形胶质细胞中，Palladin在细胞单层机械损伤后迅速上调。此外，我们还发现，在成年大鼠大脑皮层损伤后，Palladin的上调发生在类似和快速的时间过程中。这项研究的目的是回答以下问题：(1)Palladin的上调与细胞形状从星状到扁平的变化密切相关。Palladin的表达是导致这种形状变化的直接原因吗？这个问题将通过瞬时转基因技术在培养的星形胶质细胞中增加和减少Palladin的表达而得到解答。(2)Palladin在星形胶质细胞中的结合伙伴是什么，它们在机械损伤后在星形胶质细胞中是否协同上调？根据序列同源性，我们编制了一个可能直接与Palladin结合的五种蛋白质的列表。我们将在培养的星形胶质细胞中探索这些相互作用，并使用酵母双杂交筛选进行更广泛的搜索。(3)在体脑皮质损伤后，Palladin在星形胶质细胞中是否上调？双标记免疫荧光将被用来确定上调Palladin的细胞类型，并对这些细胞中伤口边缘Palladin的表达进行量化。最后，病毒载体将被用来询问当损伤部位的星形胶质细胞中Palladin的表达减少时，胶质瘢痕的形成是减弱还是被抑制。这些实验有望对星形胶质细胞对损伤刺激做出反应的基本细胞过程产生新的见解。这项研究的一个长期目标是为控制体内星形细胞反应和胶质瘢痕形成提供新的治疗方法。

DESCRIPTION (provided by applicant): Astrocytes respond to injury of the central nervous system with a dramatic change in morphology, resulting in the formation of a dense glial scar that can inhibit axonal regeneration. The molecular basis for the "reactive astrocyte" phenotype is not understood. We recently identified a novel cytoskeleton-associated protein named palladin, which plays an essential role in maintaining the actin cytoskeleton in many cell types, This proposal explores the role of palladin in the response of astrocytes to mechanical injury, both in vitro and in vivo. Our hypothesis is that palladin functions as a molecular scaffold to organize the actin cytoskeleton and promote a change in cell shape of astrocytes in response to a specific stimulus. We obtained preliminary evidence that palladin is rapidly upregulated in cultured astrocytes in response to mechanical wounding of the cell monolayer. In addition, we show that palladin upregulation occurs along a similar and rapid time-course following injury to the cerebral cortex in adult rats. The goals of the proposed research are to answer the following questions: (1) Palladin upregulation correlates closely with a change in cell shape from stellate to flattened. Is palladin expression directly responsible for this change in shape? This question will be answered using transient transfection techniques to increase and decreasepalladin expression in cultured astrocytes. (2) What are palladin's binding partners in astrocytes, and are they coordinately upregulated in astrocytes following mechanical injury? Based on sequence homologies, we have compiled a list of five proteins that are likely to bind directly to palladin. We will explore these interactions in cultured astrocytes and also search more broadly using a yeast two-hybrid screen. (3) Is palladin upregulated in astrocytes following injury to the cortex in vivo? Double-label immunofluorescence will be used to definitively identify the cell types that upregulate palladin and to quantify the expression of palladin, in these cells, at the margins of the wound. Finally, viral vectors will be used to ask if glial scar formation is attenuated or inhibited when palladin expression is reduced in astrocytes in the site of injury. These experiments are expected to produce new insights into the basic cellular processes that underlie the response of astrocytes to an injury stimulus. A long-term goal of this research is to provide new therapeutic approaches for controlling the astrocytic response and glial scar formation in vivo.