Thy-1-induced syndecan-4-dependent RhoA activation on astrocytes

描述（由申请人提供）：我们报告的数据显示，神经元-星形胶质细胞缔合中的Thy-1- 1V 23整联蛋白相互作用通过整联蛋白聚集和RhoA活化刺激星形胶质细胞形成局灶性粘连和应力纤维。局灶性粘附锚应力纤维的质膜和牵连在细胞粘附，迁移，生长。我们提交的数据表明，Thy-1需要整合素和syndecan-4通过PKC 1和RhoA依赖性途径来增加细胞与基质的粘附。有趣的是，时间RhoA激活是双相的，在5分钟时具有小的活性峰，在20分钟时具有较大的活性峰。由于RhoA据报道在PKC 1下游被激活，这项合作资助旨在确定PKC 1是否也遵循整合素和syndecan-4下游激活的双相模式，以及确定Thy-1诱导的RhoA激活所需的鸟嘌呤核苷酸交换因子（GEF）。为了测试PKC 1是否被双相激活，将使用空细胞、显性阴性构建体或药理学抑制剂研究整联蛋白或多配体蛋白聚糖的下游分子。为了评估每种途径的贡献，将使用整合素/多配体蛋白聚糖结合位点突变的Thy-1单独或组合实现细胞刺激。此外，通过FRET分析，在用Rho生物传感器转染的单细胞中跟踪Thy-1刺激后的时空Rho激活。RhoGEF将在下拉测定中使用对RhoGEF具有高亲和力的GST-17 ARhoA来鉴定.使用质谱分析沉淀的复合物将鉴定参与RhoA活化的分子。因此，将研究Thy-1诱导的星形胶质细胞形态学变化的分子机制。获得的见解应该提高我们对星形胶质细胞增生的理解，星形胶质细胞增生是脑损伤引发的一个过程，涉及星形胶质细胞的戏剧性形态变化，迁移形成胶质瘢痕和抑制人类神经元再生。这项研究将主要在智利进行，与来自智利的基思伯里奇博士合作，作为NIH资助#RO1 GM 29860 -27的延伸。该合作是使用FIRCA基金（2002年授予的RO 3 TW 006024）启动的，其中Burridge博士是PI，Lisette莱顿博士是外国研究者。公共卫生相关性：星形胶质细胞普遍存在于整个大脑中，并与神经元密切相关。在损伤时或响应于炎症，星形胶质细胞被激活、迁移、尺寸增加并参与神经胶质瘢痕的形成。在这样做时，受损区域与未受影响的组织隔离，以避免继发性病变，并为神经元再生创造了一个不允许的环境。在成年中枢神经系统中缺乏神经元再生的分子机制仍然存在争议，可能是由于多个抑制性细胞-细胞以及细胞-基质相互作用的参与。我们的兴趣集中在神经元-星形胶质细胞的相互作用，描述了第一次由我们的小组7年前，之间的Thy-1，一个高度丰富的神经元表面分子与抑制轴突生长，其报告的星形胶质细胞受体，1V 23整合素和syndecan-4。通过本文提出的研究，我们将了解控制Thy-1诱导的星形胶质细胞形态学变化的分子机制。我们的目标是更好地了解星形胶质细胞参与胶质瘢痕的形成和轴突生长的抑制。在理解轴突再生的基本事件方面已经取得了很大进展，但肯定不足以实现中枢神经系统损伤后这些结构的部分至完全恢复。目前的提案旨在更好地了解星形胶质细胞与Thy-1相互作用后发生的变化。我们实验室正在进行的另一项研究调查了1V 23整合素与Thy-1相互作用可能对神经元产生的影响。我们的研究结果表明，这种相互作用触发了两种细胞中的双向信号传导，并导致抑制分化神经元中的轴突生长，以及已经存在的过程的轴突收缩。因此，这一组的研究有望产生一个更好的理解控制神经突起生长和星形胶质细胞功能的分子机制。这些见解对一般的大脑生理学具有根本意义，并可能有助于开发更有效的促进神经再生的策略。

DESCRIPTION (provided by applicant): Our reported data show that Thy-1-1V23 integrin interaction in neuron-astrocyte association stimulates astrocytes to form focal adhesions and stress fibers by integrin clustering and RhoA activation. Focal adhesions anchor stress fibers to the plasma membrane and are implicated in cell adhesion, migration, growth. Our submitted data indicate that Thy-1 requires both integrin and syndecan-4 to increase cell adhesion to the matrix via PKC1- and RhoA-dependent pathways. Interestingly, temporal RhoA activation is biphasic with a small peak of activity at 5min and a larger one at 20min. Since RhoA is reportedly activated downstream of PKC1, this collaborative grant seeks to determine whether PKC1 also follows a biphasic mode of activation downstream of integrins and syndecan-4, as well as to identify guanine nucleotide exchange factors (GEFs) required for Thy-1-induced RhoA activation. To test whether PKC1 is activated biphasically, molecules downstream of integrins or syndecans will be studied using null cells, dominant negative constructs or pharmacological inhibitors. To assess the contribution of each pathway, cell stimulation will be achieved using Thy-1 mutated in integrin/syndecan binding sites either individually or in combination. Additionally, spatio- temporal Rho activation upon Thy-1 stimulation will be followed in single cells transfected with Rho biosensors by FRET analysis. RhoGEFs will be identified in pull- down assays using GST-17ARhoA with high affinity for RhoGEFs. The use of mass spectrometry to analyze precipitated complexes will identify molecules involved in RhoA activation. Thus, molecular mechanisms governing Thy-1-induced morphological changes in astrocytes will be studied. Insights obtained should improve our understanding of astrogliosis, a process triggered upon brain injury that involves dramatic morphological changes in astrocytes, migration to form the glial scar and inhibition of neuronal regeneration in humans. This research will be done primarily in Chile, in collaboration with Dr. Keith Burridge from UNC, as an extension of NIH grant #RO1 GM29860-27. This collaboration was initiated using FIRCA funds (RO3 TW006024 awarded in 2002) where Dr. Burridge was the PI and Dr. Lisette Leyton the Foreign Investigator. PUBLIC HEALTH RELEVANCE: Astrocytes are ubiquitously present throughout the brain and associate intimately with neurons. Upon injury or in response to inflammation, astrocytes are activated, migrate, increase in size and participate in the formation of the glial scar. In doing so, damaged areas are segregated from unaffected tissue to avoid secondary lesions and a non-permissive environment for neuronal regeneration is created. Molecular mechanisms responsible for the lack of neuronal regeneration in the adult central nervous system remain controversial, possibly due to the participation of multiple inhibitory cell-cell, as well as cell-matrix interactions. Our interest focuses on a neuron-astrocyte interaction, described for the first time by our group 7 years ago, between Thy-1, a highly abundant neuronal surface molecule associated with inhibition of neurite outgrowth, and its reported astrocyte receptors, the 1V23 integrin and syndecan-4. With the studies proposed here, we will learn about molecular mechanisms that control Thy-1- induced morphological changes in astrocytes. The goal is to understand better astrocyte participation in the formation of the glial scar and inhibition of axonal growth. Much progress has been made in understanding the basic events underlying axon regeneration, but certainly not enough to achieve partial to complete recovery of such structures following central nervous system injury. The current proposal seeks a better understanding of the changes occurring in astrocytes upon interaction with Thy-1. A different line of ongoing research in our laboratory investigates the effect that 1V23 integrin interaction with Thy-1 might have in neurons. Our results indicate that this interaction triggers bidirectional signaling in both cells and leads to inhibition of neurite outgrowth in differentiating neurons, as well as axonal retraction of already existing processes. Thus, studies of this group are expected to yield a better understanding of molecular mechanisms controlling neurite outgrowth and astrocyte function. Such insights are of fundamental interest to brain physiology in general and may help in the development of more effective strategies to promote nerve regeneration.