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

Thy-1 诱导星形胶质细胞上的 syndecan-4 依赖性 RhoA 激活

• 批准号: 7806407
• 负责人: Lisette Leyton
• 金额: $ 3.25万
• 依托单位: UNIVERSITY OF CHILE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-08 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7806407
• 关键词: 1,2-diacylglycerol; Address; Adherens Junction; Adhesions; Adult; Affect; Affinity; Antibodies; Area; Astrocytes; Award; Back; Binding; Binding Sites; Biological Assay; Biosensor; Brain; Brain Injuries; C-terminal; Cadherin Domain; Cadherins; Cell Adhesion; Cell Surface Proteins; Cell membrane; Cell-Cell Adhesion; Cells; Chile; Cicatrix; Collaborations; Complex; Consensus Sequence; Coupled; Data; Data Reporting; Development; Diglycerides; Dominant-Negative Mutation; Employee Strikes; Environment; Equipment; Event; Extracellular Matrix; Extracellular Matrix Proteins; Family; Family member; Fibronectins; Fluorescence Resonance Energy Transfer; Focal Adhesions; Funding; Future; Goals; Grant; Growth; Guanine Nucleotide Exchange Factors; Heparan Sulfate Proteoglycan; Heparin Binding; Human; Immunoblotting; Inflammation; Injury; Integrin Signaling Pathway; Integrins; Intercellular Junctions; Isometric Exercise; Laboratories; Learning; Ligation; Maps; Mass Spectrum Analysis; Measures; Mediating; Methodology; Modification; Molecular; Molecular Biology Techniques; Mutate; Natural regeneration; Nature; Nerve Regeneration; Nervous system structure; Neuraxis; Neurites; Neurons; Nucleotides; Null Lymphocytes; PDZ protein; Pathway interactions; Persons; Phosphorylation; Phosphotransferases; Physiology; Precipitation; Process; Protein Tyrosine Kinase; Proteins; Proteoglycan; Recombinant Proteins; Recombinants; Recovery; Recruitment Activity; Relative (related person); Reporting; Research; Research Personnel; Secondary Lesion; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; Stress Fibers; Structure; Surface; System; Technology; Testing; Tight Junctions; Time; Tissues; United States National Institutes of Health; Work; astrogliosis; axon growth; axon regeneration; cell behavior; cell motility; central nervous system injury; flexibility; graduate student; improved; inhibitor/antagonist; insight; interest; member; migration; mutant; novel; parent grant; physical state; public health relevance; receptor; research study; response; rho; scaffold; syndecan; syndecan-4

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.

描述（申请人提供）：我们报道的数据表明，神经元-星形胶质细胞关联中的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粘连和应力纤维。局灶黏附将应力纤维锚定在质膜上，并与细胞黏附、迁移和生长有关。我们提交的数据表明，Thy-1需要整合素和syndecan-4来通过PKC1和rhoa依赖途径增加细胞对基质的粘附。有趣的是，颞叶RhoA的激活是双相的，在5min时出现一个小的活动峰，在20min时出现一个大的活动峰。由于RhoA在PKC1下游被激活，因此该合作资助旨在确定PKC1是否也遵循整合素和syndecan-4下游的双相激活模式，以及确定y-1诱导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，将使用空细胞、显性阴性构建体或药理抑制剂研究整合素或syndecans下游的分子。为了评估每种途径的作用，将使用整合素/syndecan结合位点突变的Thy-1单独或联合进行细胞刺激。此外，通过FRET分析，在转染了Rho生物传感器的单细胞中，Thy-1刺激后Rho的时空激活将被遵循。rhogef将在下拉试验中使用对rhogef具有高亲和力的GST-17ARhoA进行鉴定。使用质谱分析沉淀络合物将识别参与RhoA活化的分子。因此，thy -1诱导星形胶质细胞形态学改变的分子机制将得到进一步研究。获得的见解将提高我们对星形胶质细胞形成的理解，这是一个由脑损伤引发的过程，涉及星形胶质细胞的剧烈形态变化，形成胶质疤痕的迁移和人类神经元再生的抑制。这项研究将主要在智利进行，与UNC的Keith Burridge博士合作，作为NIH拨款#RO1 GM29860-27的延伸。这项合作是由FIRCA基金发起的（2002年颁发的RO3 TW006024）， Burridge博士是PI， Lisette Leyton博士是外国调查员。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。在损伤或炎症反应时，星形胶质细胞被激活、迁移、大小增加并参与胶质瘢痕的形成。这样，受损区域与未受影响的组织分离，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。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，可能是由于多种抑制细胞-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与星形胶质细胞之间的相互作用，这是我们的小组在7年前首次描述的，在Thy-1（一种与神经突生长抑制相关的高度丰富的神经元表面分子）与其报道的星形胶质细胞受体，1V23整合素和syndecan-4之间。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。目的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些结构的部分或完全恢复。目前的建议是为了更好地了解星形胶质细胞在与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。我们的研究结果表明，这种相互作用在两个细胞中触发双向信号，并导致分化神经元中神经突生长的抑制，以及已经存在的过程的轴突缩回。因此，这一组的研究有望更好地理解控制神经突生长和星形胶质细胞功能的分子机制。这些见解对大脑生理学具有根本性的意义，可能有助于开发更有效的策略来促进神经再生。