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

描述(申请人提供)：我们的报告数据显示，神经元-星形胶质细胞联合中的Thy-1-1V23整合素相互作用通过整合素聚集和RhoA激活刺激星形胶质细胞形成局灶性粘连和应力纤维。局灶性粘连将应力纤维锚定在质膜上，与细胞的黏附、迁移、生长有关。我们提交的数据表明，Thy-1需要整合素和Syndecan-4来增加细胞与基质的黏附，这是通过依赖PKC1和RhoA的途径实现的。有趣的是，瞬时RhoA的激活是双相的，在5min有一个小的激活峰值，在20min有一个较大的激活峰值。由于据报道，RhoA是在PKC1下游被激活的，所以这项合作资助旨在确定PKC1是否也遵循整合素和Syndecan-4下游的两相激活模式，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PKC1是否被双相激活，整合素或合并素下游的分子将使用零细胞、显性负结构或药理抑制剂进行研究。为了评估每条途径的贡献，将使用整合素/Syndecan结合位点上突变的Thy-1单独或组合实现细胞刺激。此外，通过FRET分析，在Thy-1刺激下，Rho在单个细胞中的时空激活将被转移到Rho生物传感器中。RhoGEF将通过GST-17ARhoA与RhoGEF的高亲和力在下拉试验中进行鉴定。使用质谱仪分析沉淀的络合物将识别参与RhoA活化的分子。因此，调控Thy-1诱导星形胶质细胞形态变化的分子机制将被研究。所获得的见解应该会提高我们对星形胶质细胞增生症的理解，这是一种由脑损伤引发的过程，涉及星形胶质细胞的戏剧性形态变化，迁移形成胶质疤痕，以及抑制人类神经元的再生。这项研究将主要在智利进行，与北卡罗来纳大学的基思·布里奇博士合作，作为NIH#RO1 GM29860-27号拨款的延伸。这项合作是利用FIRCA Funds(2002年授予的R03 TW006024)发起的，布里奇博士担任私人助理，利塞特·莱顿博士担任外国调查员。与公共健康相关：星形胶质细胞在大脑中无处不在，并与神经元密切相关。在损伤或炎症反应中，星形胶质细胞被激活、迁移、增大，并参与胶质瘢痕的形成。在这样做的过程中，受损区域与未受影响的组织被隔离，以避免继发性损害，并为神经元再生创造了一个不允许的环境。成人中枢神经系统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-细胞的参与，以及细胞-基质的相互作用。我们的兴趣集中在神经元和星形胶质细胞之间的相互作用，这是我们团队7年前首次描述的，Thy-1是一种高度丰富的神经元表面分子，与抑制轴突生长有关，与其已报道的星形胶质细胞受体1V23整合素和Syndecan-4之间的相互作用。通过这里提出的研究，我们将了解控制Thy-1诱导星形胶质细胞形态变化的分子机制。目的是更好地了解星形胶质细胞在胶质瘢痕形成和抑制轴突生长中的作用。在了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种结构的部分或完全恢复。目前的建议试图更好地理解星形胶质细胞与Thy-1相互作用时发生的变化。我们实验室正在进行的另一项研究调查了1V23整合素与Thy-1相互作用可能对神经元产生的影响。我们的结果表明，这种相互作用在两个细胞中触发双向信号，并导致抑制分化神经元中的轴突生长，以及已经存在的突起的轴突回缩。因此，对这一组的研究有望更好地理解控制轴突生长和星形胶质细胞功能的分子机制。这些见解对整个大脑生理学具有根本意义，并可能有助于开发更有效的策略来促进神经再生。

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.