Role of Palladin in Regulating Astrocytes

描述（由申请人提供）：星形胶质细胞对中枢神经系统损伤的反应是形态的急剧变化，导致致密胶质瘢痕的形成，可以抑制轴突再生。“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名为palladin的新型细胞骨架相关蛋白，它在维持许多细胞类型的肌动蛋白细胞骨架中起着至关重要的作用，本研究旨在探讨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。我们的假设是钯作为一种分子支架来组织肌动蛋白细胞骨架，并促进星形胶质细胞在特定刺激下细胞形状的变化。我们获得的初步证据表明，在培养的星形胶质细胞中，钯素在细胞单层机械损伤的反应中迅速上调。此外，我们发现，在成年大鼠大脑皮层损伤后，帕拉丁素的上调发生在一个类似的快速时间过程中。提出的研究目标是回答以下问题：(1)帕拉丁上调与细胞形状从星状到扁平的变化密切相关。帕拉丁人的表达是否直接导致了这种形状的变化？这个问题将用瞬时转染技术来增加和减少epalladin在培养的星形胶质细胞中的表达来回答。(2)什么是palladin在星形胶质细胞中的结合伙伴，它们是否在机械损伤后的星形胶质细胞中协同上调？基于序列同源性，我们编制了一个可能直接与palladin结合的5个蛋白的列表。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)体内皮层损伤后星形胶质细胞中钯素是否上调？双标记免疫荧光将用于明确识别上调帕拉丁的细胞类型，并量化这些细胞在伤口边缘的帕拉丁表达。最后，病毒载体将用于研究损伤部位星形胶质细胞中钯素表达降低是否会减弱或抑制胶质瘢痕的形成。这些实验有望对星形胶质细胞对损伤刺激反应的基本细胞过程产生新的见解。本研究的长期目标是为体内控制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.