Variation in Neuroligin Concentration and Presynaptic Functional Development

描述（由申请人提供）：精神分裂症和自闭症是毁灭性的，复杂的大脑疾病，其原因知之甚少。广泛的遗传学证据将自闭症和精神分裂症与细胞粘附分子神经配素的突变联系起来。疾病相关突变改变突触后膜的神经连接素水平。异常的神经连接素水平与不成熟的突触形成相关，包括释放神经递质和回收突触囊泡的机制的不成熟。这些发现提高了通过靶向囊泡释放和再循环机制来影响成熟的突触前表型来治疗自闭症和精神分裂症的可能性。我们将使用突触囊泡胞吐的荧光标记物来研究不同神经配素浓度对突触发育和功能的影响。为了模拟异常的神经连接素浓度，我们将神经连接素连接到大约突触大小的微岛中的盖玻片上，并在其上培养解离的海马神经元。以往的研究表明，神经连接素足以诱导神经元形成突触前终扣。我们的初步实验表明，神经元确实形成突触前专门化的神经胶质蛋白图案的玻璃。与目前的技术不同，这种新的细胞培养基质使我们能够精确地控制“突触后”神经连接素的水平，并解开神经连接素对突触前和突触后发育的影响。对玻璃形成的突触前终端的几何形状将使我们能够应用高分辨率全内反射（TIRF）显微镜，以及在相同的终端的落射荧光。本文提出的工作将是TIRF首次应用于成熟的小中枢神经元的突触前区室。TIRF成像提供的突触前末梢的详细视图对于理解正常和病理性突触前功能具有广泛的意义。这里描述的技术可以进一步发展，以快速筛选针对异常突触发育，囊泡回收和神经递质释放的治疗药物。

DESCRIPTION (provided by applicant): Schizophrenia and autism are devastating, complex brain disorders whose causes are poorly understood. Extensive genetic evidence links autism and schizophrenia to mutations in the cell adhesion molecule neuroligin. Disease-linked mutations alter neuroligin levels at the postsynaptic membrane. Abnormal neuroligin levels are associated with immature synapse formation, including immaturity of mechanisms to release neurotransmitter and recycle synaptic vesicles. These findings raise the possibility of treating autism and schizophrenia by targeting mechanisms of vesicular release and recycling to effect the mature presynaptic phenotype. We will use fluorescent markers of synaptic vesicle exocytosis to study the effect of varying neuroligin concentrations on synapse development and function. To model abnormal neuroligin concentration, we will attach neuroligin to coverslips in microislands roughly the size of a synapse and culture dissociated hippocampal neurons on them. Previous studies have shown that neuroligin is sufficient to induce neurons to form presynaptic boutons. Our preliminary experiments suggest that neurons indeed form presynaptic specializations on neuroligin-patterned glass. Unlike current technologies, this novel cell culture substrate allows us to control "postsynaptic" neuroligin levels precisely and disentangle neuroligin's effects on presynaptic and postsynaptic development. The geometry of the presynaptic terminals that form against the glass will allow us to apply high-resolution total internal reflection (TIRF) microscopy as well as epifluorescence at the same terminals. The work proposed here would be the first application of TIRF to the presynaptic compartment of maturing small central neurons. The detailed view of the presynaptic terminal offered by TIRF imaging has broad implications for understanding normal and pathological presynaptic function. The technology described here could be further developed to rapidly screen therapeutic agents that target abnormal synapse development, vesicle recycling, and neurotransmitter release.