Variation in Neuroligin Concentration and Presynaptic Functional Development

Neuroligin 浓度的变化和突触前功能发育

• 批准号: 8702366
• 负责人: Susan M. Voglmaier
• 金额: $ 19.7万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-10 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8702366
• 关键词: Affect; Autistic Disorder; Binding; Biological Assay; Brain; Brain Diseases; Cell Adhesion Molecules; Cell Culture Techniques; Cells; Complex; Development; Developmental Process; Disease; Drug Targeting; Endocytosis; Exocytosis; Fluorescence Microscopy; Future; Genetic; Geometry; Glass; Goals; Hippocampus (Brain); Image; Kinetics; Life; Link; Measures; Methods; Microscopy; Modeling; Molecular; Mutation; Neurobiology; Neuroglia; Neurons; PHluorin; Pattern; Pharmaceutical Preparations; Phenotype; Plant Resins; Population; Postsynaptic Membrane; Presynaptic Terminals; Printing; Property; Rattus; Recycling; Regulation; Reporter; Research; Resolution; Schizophrenia; Simulate; Stem cells; Surface; Synapses; Synaptic Vesicles; Technology; Testing; Therapeutic Agents; Time; Variant; Vesicle; Work; base; effective therapy; inhibitor/antagonist; inhibitory neuron; mutant; neuropsychiatry; neurotransmitter release; new technology; novel; postsynaptic; presynaptic; public health relevance; research study; screening; synaptogenesis; time use; transmission process

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.

描述（由申请人提供）：精神分裂症和自闭症是毁灭性的、复杂的脑部疾病，其原因尚不清楚。大量的遗传证据表明自闭症和精神分裂症与细胞粘附分子神经胶质蛋白的突变有关。与疾病相关的突变会改变突触后膜的神经胶质素水平。异常的神经信号素水平与不成熟的突触形成有关，包括释放神经递质和回收突触小泡的机制不成熟。这些发现提出了通过针对囊泡释放和再循环机制来影响成熟突触前表型来治疗自闭症和精神分裂症的可能性。我们将使用突触小泡胞吐作用的荧光标记来研究不同的 Neuroligin 浓度对突触发育和功能的影响。为了模拟异常的 Neuroligin 浓度，我们将 Neuroligin 附着在突触大小的微岛上的盖玻片上，并在其上培养分离的海马神经元。先前的研究表明，neuroligin 足以诱导神经元形成突触前神经元。我们的初步实验表明，神经元确实在神经肽图案玻璃上形成突触前特化。与现有技术不同，这种新型细胞培养基质使我们能够精确控制“突触后”神经胶质素水平，并解开神经胶质素对突触前和突触后发育的影响。靠着玻璃形成的突触前末端的几何形状将使我们能够在同一末端应用高分辨率全内反射（TIRF）显微镜以及落射荧光。这里提出的工作将是 TIRF 首次应用于成熟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开发，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。