INTEGRIN LIGANDS IN SYNAPTIC PLASTICITY

突触可塑性中的整合素配体

• 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
• 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

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 A.具体目标 资助提案的具体目的没有重大变化。 B.研究和结果 特定目的1是测试CA1整联蛋白配体。为了找到整联蛋白配体，我们继续使用海马神经突触体作为突触材料的资源。在当前预算年度，我们的交联实验和细胞印迹实验仍在继续。我们想从更多实验中获得结果，以验证数据是否可靠地重现。细胞印迹实验揭示了四个支持粘附的频段。通过通过聚丙烯酰胺凝胶电泳来解决海马，神经气体，将蛋白质转移到膜上，并使用膜作为底层来进行印迹。我们测试了几辆车辆以将细胞与膜孵育，并找到了2种有用的方法。我们报告的一种是使用注射器与膜孵育细胞。另一种是专为免疫印迹设计的设备，我们用于活细胞。两辆车都以类似的效率运行。 频段确定在大约50、75、100和150 kDa的位置迁移。 50 kDa区域的质谱分析鉴定了许多蛋白质。 50 kDa区域中的蛋白质，一个或多个支持细胞附着的附着。我们正在提交更多用于质谱分析的样本，以查看是否存在从不同制剂的神经突触细胞分离的50 kDa区域的分子上复制细胞粘附，并在75、100和150 kDa区域中测试配体鉴定。 特定AIM 2测试整合素配体位置的实验。使用免疫传输电子显微镜，我们在CA1中定位了抗整合蛋白抗体。数据表明整联蛋白在突触。我们还在测试LFS和HFS海马切片中荧光团标记的GRGDSP整合蛋白配体肽的位置。我们的共聚焦图像表明配体位于Ca1顶端树突附近。现在，我们正在测试与肌动蛋白和突触素蛋白共定位。 特定AIM 3中的实验将测试配体堵塞时对LTP的影响。我们已经在培养的细胞上测试了RNA1寡核苷酸，以查看是否可以击倒整联蛋白。使用标准转染技术，我们通过定量PCR和免疫印迹分析获得了整联蛋白表达的降低60％。这有助于我们为通过质谱识别的敲低配体奠定了基础。敲低是在CA1 LTP中测试配体角色的好方法。抗体形抗体（如果有）将用作互补方法。 C.意义 突触可塑性的分子过程是复杂的，并由许多不同细胞外，细胞表面和细胞内分子调节。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CA很重要！突触事件。在海马切片中，抗整合蛋白抗体和整联蛋白配体肽拮抗了LTP的维持。有趣的是，在海马切片中，HFS触发了分泌囊泡到细胞表面的整联蛋白的易位。易位发生在LTP诱导后的1-2分钟内，因此预期整联蛋白表达有望促进细胞骨架的重要变化，突触和附近突触的质膜形状，并激活细胞内信号通路。尽管整联蛋白配体在其他系统中是已知的，但整联蛋白配体的身份和位置对于维持Ca1 LTP的维持很重要。 我们的超微结构数据表明整联蛋白处于突触，质谱分析发现了神经突触体粘附蛋白。我们目前正在测试发现是否可再现。 如果发现该发现并找到配体，它将帮助我们了解维护LTP的何时何地发生信号操作。新信息将填补我们目前的知识中的空白，并有望帮助我们了解神经退行性疾病的基础过程。