INTEGRIN LIGANDS IN SYNAPTIC PLASTICITY

突触可塑性中的整合素配体

基本信息

批准号：
7715339
负责人：
Richard Gordon LeBaron
金额：
$ 13.13万
依托单位：
UNIVERSITY OF TEXAS SAN ANTONIO
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-01 至 2009-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7715339
关键词：
Actins Adhesions Antibodies Apical Biochemical Genetics Budgets Cell Adhesion Cell membrane Cell surface Cell-Matrix Junction Cells Chromosome Pairing Complex Computer Retrieval of Information on Scientific Projects Database Cultured Cells Cytoskeleton Data Dendrites Event Fostering Funding Gel Grant Hippocampus (Brain)Image Immunoblotting Incubated Institution Integrins Knowledge Label Life Ligands Location Maintenance Mass Spectrum Analysis Membrane Methods Molecular Neurodegenerative Disorders Numbers Oligonucleotides Peptides Polyacrylamide Gel Electrophoresis Polymerase Chain Reaction Positioning Attribute Preparation Process Proteins RNA I Reporting Research Research Personnel Resources Role Sampling Secretory Vesicles Shapes Signal Pathway Signal Transduction Slice Source Staging Standards of Weights and Measures Supporting Cell Synapses Synaptic plasticity Synaptophysin Syringes System Techniques Testing Transfection Transmission Electron Microscopy United States National Institutes of Health Work crosslink design extracellular fluorophore glycyl-arginyl-glycyl-aspartyl-seryl-proline knock-down numb protein receptor research study

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A. Specific Aims There were no significant changes in the Specific Aims of the funded proposal. B. Studies and Results Specific Aim 1 was to test for CA1 integrin ligands. To find integrin ligands we have continued to use hippocampal neurosynaptosomes as a resource for synaptic material. Our cross-linking experiments and cell blot experiments continued in the current budget year. We wanted to obtain results from more experiments in order to verify whether the data were reliably reproducible. The cell blot experiments have revealed four bands that support adhesion. The blot is conducted by resolving hippocampal, neurosynaptosomes by polyacrylamide gel electrophoresis, transferring the proteins to a membrane, and use of the membrane as a substratum. We tested several vehicles to incubate cells with the membranes, and found 2 useful methods. One, which we reported, was the use of syringes to incubate cells with the membrane. The other was a apparatus designed specifically for immunoblots, which we used for living cells. Both vehicles worked with similar efficiency. The bands identified migrate at positions corresponding to approximately 50, 75, 100 and 150 kDa. Mass spectrometry analysis of the 50 kDa region identified a number of proteins. Of the proteins in the 50 kDa region, one or more support cell attachment reliably. We are submitting more samples for mass spectrometry analysis in order to see whether there are cell adhesion is reproduced on molecules from the 50 kDa region isolated from neurosynaptosomes of different preparations, and test for ligand identity in the 75, 100, and 150 kDa regions of the gel. The experiments in Specific Aim 2 test for the location of integrin ligands. Using immuno-transmission electron microscopy we have located anti-integrin antibody in CA1. The data indicate integrins are at synapses. We are additionally testing for the location of a fluorophore-labeled GRGDSP integrin ligand peptide in LFS and HFS hippocampal slices. Our confocal images indicate that the ligand is located near CA1 apical dendrites. We are now testing for colocalizations with actin and synaptophysin. The experiments in Specific Aim 3 will test for effects on LTP when ligand in blocked. We have tested RNA1 oligonucleotides on cultured cells to see whether we can knock down integrins. Using standard transfection techniques we achieved a 60% reduction in integrin expression, as accessed by quantitative PCR and immunoblot analyses. This helps us set the stage to knockdown ligand identified by mass spectrometry. The knockdowns are a good approach to test for ligand roles in CA1 LTP. Anti-ligand antibodies, if available, will serve as a complementary approach. C. Significance Molecular processes underlying synaptic plasticity are complex and regulated by a number of different extracellular, cell-surface and intracellular molecules. There is compelling genetic and biochemical evidence that the cell-surface integrin receptors are important for CA! synaptic events. In hippocampal slices, anti-integrin antibodies and integrin ligand peptides antagonized the maintenance of LTP. Interestingly, in hippocampal slices, HFS triggers translocation of integrins that are in secretory vesicles to the cell surface. The translocation occurs within 1-2 minutes after LTP induction, so increased integrin expression is expected to foster important changes in the cytoskeleton, the shape of plasma membranes at and near synapses, and activate intracellular signaling pathways. Although integrin ligands are known in other systems, the identity and location of integrin ligand important for the maintenance of CA1 LTP is not clear. Our ultrastructural data indicates that integrins are at synapses, and mass spectrometry analysis found neurosynaptosome adhesion proteins. We are presently testing whether the findings are reproducible. If the finding is verified and ligand found, it will help us understand the when and where the signaling actions occur for the maintenance of LTP. The new information will fill in gaps in our present knowledge, and is expected to help us understand processes underlying neurodegenerative disorders.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目和研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是对于中心来说，它不一定是研究者的机构。 A. 具体目标资助提案的具体目标没有重大变化。 B. 研究和结果具体目标 1 是测试 CA1 整合素配体。为了寻找整合素配体，我们继续使用海马神经突触体作为突触材料的资源。我们的交联实验和细胞印迹实验在本预算年度继续进行。我们希望从更多的实验中获得结果，以验证数据是否可靠可重复。细胞印迹实验揭示了四个支持粘附的条带。通过聚丙烯酰胺凝胶电泳解析海马神经突触体，将蛋白质转移到膜上，并使用膜作为基质来进行印迹。我们测试了几种用膜孵育细胞的载体，发现了 2 种有用的方法。我们报道的其中一种是使用注射器将细胞与膜一起孵育。另一个是专门为免疫印迹设计的装置，我们将其用于活细胞。两种车辆的工作效率相似。识别出的条带在对应于大约 50、75、100 和 150 kDa 的位置处迁移。对 50 kDa 区域的质谱分析鉴定出许多蛋白质。在 50 kDa 区域的蛋白质中，一种或多种能够可靠地支持细胞附着。我们正在提交更多样品进行质谱分析，以观察从不同制备物的神经突触体中分离出的 50 kDa 区域的分子是否复制了细胞粘附，并测试了 75、100 和 150 kDa 区域的配体身份。凝胶。具体目标 2 中的实验测试整合素配体的位置。使用免疫透射电子显微镜，我们在 CA1 中找到了抗整合素抗体。数据表明整合素位于突触处。我们还在 LFS 和 HFS 海马切片中测试荧光团标记的 GRGDSP 整合素配体肽的位置。我们的共焦图像表明配体位于 CA1 顶端树突附近。我们现在正在测试与肌动蛋白和突触素的共定位。具体目标 3 中的实验将测试配体被阻断时对 LTP 的影响。我们在培养细胞上测试了 RNA1 寡核苷酸，看看是否可以敲除整合素。通过定量 PCR 和免疫印迹分析得出，使用标准转染技术，我们实现了整合素表达降低 60%。这有助于我们为通过质谱鉴定的敲低配体奠定基础。敲低是测试 CA1 LTP 中配体作用的好方法。抗配体抗体（如果有）将作为一种补充方法。 C、意义突触可塑性的分子过程很复杂，并受到许多不同的细胞外、细胞表面和细胞内分子的调节。有令人信服的遗传和生化证据表明细胞表面整合素受体对于 CA 很重要！突触事件。在海马切片中，抗整合素抗体和整合素配体肽拮抗 LTP 的维持。有趣的是，在海马切片中，HFS 触发分泌囊泡中的整合素易位到细胞表面。易位发生在 LTP 诱导后 1-2 分钟内，因此整合素表达的增加预计将促进细胞骨架、突触处和突触附近质膜形状的重要变化，并激活细胞内信号传导途径。尽管整合素配体在其他系统中是已知的，但对于维持 CA1 LTP 重要的整合素配体的身份和位置尚不清楚。我们的超微结构数据表明整合素位于突触，质谱分析发现神经突触体粘附蛋白。我们目前正在测试研究结果是否可重复。如果这一发现得到验证并找到配体，将有助于我们了解维持 LTP 的信号传导作用何时何地发生。新信息将填补我们现有知识的空白，并有望帮助我们了解神经退行性疾病的潜在过程。