INTEGRIN LIGANDS IN SYNAPTIC PLASTICITY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 A.具体目标 获得资助的提案的具体目标没有重大变化。 B.研究和成果 具体目的1是检测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小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。我们想从更多的实验中获得结果，以验证数据是否可靠地重现。细胞印迹实验显示了四条支持黏附的条带。印迹是通过聚丙烯酰胺凝胶电泳法分辨海马和神经突触体来进行的，将蛋白质转移到膜上，并以膜为底物。我们测试了几种用膜培养细胞的方法，发现了两种有用的方法。一种是使用注射器将细胞与膜孵化，这是我们报道的。另一个是专门为免疫印迹设计的仪器，我们用它来处理活细胞。这两辆车的工作效率相似。 确定的条带迁移在大约50、75、100和150 kDa的位置。对50 kDa区域进行了质谱分析，确定了一些蛋白质。在50 kDa区域的蛋白质中，有一个或多个蛋白质可靠地支持细胞附着。我们正在提交更多的样本进行质谱分析，以了解从不同制剂的神经突触体内分离的50 kDa区域的分子上是否存在细胞黏附，并测试凝胶的75、100和150 kDa区域的配体特性。 特异性实验2检测整合素配体的定位。利用免疫透射电子显微镜，我们在CA1中定位了抗整合素抗体。数据表明整合素位于突触上。此外，我们还在LFS和HFS海马片中检测荧光团标记的GRGDSP整合素配基多肽的位置。我们的共聚焦图像表明，该配体位于CA1顶端树突附近。我们现在正在测试肌动蛋白和突触素的共同定位。 在特定目标3的实验将测试当配体被封闭时对LTP的影响。我们已经在培养细胞上测试了RNA1寡核苷酸，看看我们是否可以击倒整合素。通过定量聚合酶链式反应和免疫印迹分析，使用标准的转基因技术，我们实现了整合素表达的60%的降低。这有助于我们为通过质谱学鉴定击倒配体奠定基础。敲除是测试CA1LTP中配体角色的好方法。如果有抗配体抗体，将作为一种补充方法。 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对CA非常重要！突触事件。在海马片中，抗整合素抗体和整合素配基多肽拮抗LTP的维持。有趣的是，在海马片中，HFS触发分泌囊泡中的整合素移位到细胞表面。这种转位发生在LTP诱导后的1-2分钟内，因此整合素表达的增加有望促进细胞骨架、突触及其附近质膜形状的重要变化，并激活细胞内信号通路。虽然整合素配体在其他系统中是已知的，但对维持CA1 LTP至关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，质谱分析发现了神经突触体黏附蛋白。我们目前正在测试这些发现是否可以重现。 如果这一发现得到证实，并找到了配体，这将有助于我们了解LTP维持的信号转导作用发生的时间和地点。这些新信息将填补我们目前知识的空白，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.