INTEGRIN LIGANDS IN SYNAPTIC PLASTICITY

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 A.具体目标 获拨款建议的具体目的并无重大改变。 B。研究和结果 具体目标1是检测CA 1整联蛋白配体。为了找到整合素配体，我们继续使用海马神经突触体作为突触材料的来源。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，以验证数据是否具有可靠的重现性。细胞印迹实验揭示了支持粘附的四条带。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，并使用膜作为基质进行印迹。我们测试了几种载体与膜一起孵育细胞，并发现了2种有用的方法。其中之一，我们报告，是使用注射器与膜孵育细胞。另一个是专门为免疫印迹设计的仪器，我们用它来检测活细胞。两辆车的工作效率相似。 鉴别的条带在对应于约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。在50 kDa区域中的蛋白质中，一个或多个支持细胞可靠地附着。我们正在提交更多的样品进行质谱分析，以了解是否有细胞粘附在从不同制剂的神经突触体分离的50 kDa区域的分子上复制，并测试凝胶的75，100和150 kDa区域的配体身份。 特异性目标2中的实验测试整联蛋白配体的位置。使用免疫透射电镜，我们已经定位在CA 1的抗整合素抗体。数据表明整合素在突触处。我们还测试了荧光团标记的GRGDSP整合素配体肽在LFS和HFS海马切片中的位置。我们的共聚焦图像表明，配体位于CA 1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。 特定目标3中的实验将检测配体被阻断时对LTP的影响。我们已经在培养的细胞上测试了RNA 1寡核苷酸，看看我们是否可以敲低整合素。使用标准的转染技术，我们实现了60%的整合素表达减少，通过定量PCR和免疫印迹分析。这有助于我们为敲除质谱鉴定的配体奠定基础。敲除是测试配体在CA 1 LTP中作用的良好方法。抗配体抗体（如果可用）将作为补充方法。 C.意义 突触可塑性背后的分子过程是复杂的，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是重要的CA！突触事件在海马脑片中，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。易位在LTP诱导后1-2分钟内发生，因此预期增加的整合素表达促进细胞骨架、突触处和附近的质膜形状的重要变化，并激活细胞内信号传导途径。虽然在其他系统中已知整合素配体，但对于维持CA 1 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，整合素是在突触，质谱分析发现神经突触体粘附蛋白。我们目前正在测试这些发现是否具有重现性。 如果这一发现得到证实并找到配体，将有助于我们了解LTP维持的信号传导作用发生的时间和地点。新的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。

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.