Identification and characterization of modulators affecting Amyloid precursor protein (APP) family members synaptogenic activity

除了在阿尔茨海默病病理中的基础作用外，淀粉样前体蛋白(APP)在突触中也具有重要的生理功能。有趣的是，实际数据表明，APP及其同系物APLP1和APLP2在不同的突触过程中共同作用，在此过程中分泌的(SAPP)和全长APP实现不同的功能。因此，在APP/APL2基因敲除小鼠中观察到的突触形成和LTP缺陷只能部分地被SAP拯救。我们和其他人已经证明了全长APP、APLP1和APLP2可以跨向二聚化，并可能表明APP在与原代神经元共同培养的HEK293细胞中的表达有效地促进了接触轴突的突触发生，类似于其他突触黏附分子，如神经连接蛋白/Neurexins。在基于纯化的APP/APLP体外反式二聚的预筛选中，我们观察到包括神经元和神经胶质细胞在内的不同类型的细胞分泌不同的蛋白质组分来调节APP的反式二聚化。在第一次生化分析中，我们可以确定影响APP和APLP1反式定向相互作用的调节剂之一。在本申请的背景下，我们打算筛选APP/APLP反式相互作用的额外调节器，并分析它们在APP/APLP突触形成中的功能。为此，我们打算通过长期体内双光子活细胞成像，分析这些调节剂对APP/APLPs介导的原代神经元突触形成、混合共培养实验中半突触的影响以及对突触动力学的影响。在这种情况下，我们也将使用不同的APP、APLP1和APLP2单基因或双基因敲除的神经元培养。综上所述，这些研究将有助于加深我们对APP/APLP在突触调节中与其他突触蛋白相互作用的生理功能的理解。

Besides its fundamental role in Alzheimer Disease pathology, the Amyloid precursor protein (APP) has important physiological functions at the synapse. Interestingly, actual data suggest that APP and its homologues APLP1 and APLP2 act together in different synaptic processes and in doing so secreted (sAPP) and full-length APP fulfill different functions. So deficits in synapse formation and LTP observed in APP/APL2 knockout mice can only partly be rescued by sAPP. We and others have shown that full-length APP, APLP1 and APLP2 can dimerize in trans-orientation and could show that expression of APP in HEK293 cells co-cultured with primary neurons potently promotes synaptogenesis in contacting axons, similar to other synaptic adhesion molecules, such as Neuroligins/Neurexins. In a pre-screen based on in vitro trans-dimerization of purified APP/APLPs we observed that different cell types, including neurons and glia cells secrete diverse protein components modulating APP trans-directed dimerization. In first biochemical analysis we could identify one of those modulators affecting trans-directed interaction of APP and APLP1. In context of this application we intend to screen for additional modulators of APP/APLPs trans-interaction and like to analyze their function in APP/APLPs synapse formation. For this purpose we intend to analyze the impact of those modulators on APP/APLPs-mediated synapse formation in primary neurons, on hemi-synapses in the mixed co-culture assay and on synapse dynamics by long-term in vivo two-photon live cell imaging. In this context we will also use neuronal cultures from different single or double knockouts of APP, APLP1 and APLP2. Together these studies will help to increase our understanding of APP/APLPs physiological function in interplay with other synaptic proteins in synapse modulation.