High-throughput screening platform for discovery of fluorescent synaptic markers

描述（申请人提供）：突触是大脑中神经回路的基本组成部分。人们普遍认为，许多神经精神疾病的病因是突触功能障碍，包括突触形成异常或突触传递缺陷。然而，很少有工具可用于成像突触密度和功能在活的哺乳动物的大脑。虽然病毒传递基因编码探针（如荧光蛋白、通道视紫红质）被广泛用于啮齿动物大脑突触的实时成像/功能分析，但这种方法具有高度侵入性，需要专门的技术和试剂，并且在非人类灵长类动物中取得的成功有限，更不用说人类了。因此，我们提出了一种基于有机小分子对天然突触成分的分子识别的替代方法，从而促进它们对突触的选择性定位。这种“化学”方法有可能通过非侵入性给药途径将突触标记物的使用从啮齿动物模型扩展到非人类灵长类动物和人类。这种突触标记物还可以作为分子靶向装置，为活体哺乳动物大脑中的突触传递传感器和治疗药物，为神经科学研究以及神经精神疾病的诊断和治疗提供变革性工具。作为实现这些长期目标的第一步，我们建议在本应用程序中开发一个高通量筛选（HTS）平台，用于发现小分子荧光突触标记物。我们已经获得了一个基于不同荧光团结构核心的约8000种新型荧光化合物的文库，涵盖了广泛的结构和光谱范围。这种独特的资源是发现和开发突触标记物的宝贵工具。在Aim 1中，我们将使用96孔板格式的培养皮层神经元开发HTS试验。突触标记将基于标记突触前钮扣的荧光蛋白（FP）标记的突触囊泡相关蛋白的共定位进行评估。筛选、成像、数据挖掘和命中选择协议将通过约1,000-2,000个荧光化合物的初步筛选进行优化。在Aim 2中，我们将使用Aim 1中优化的方案筛选整个约8000种荧光染料库，并鉴定标记突触的击中化合物。我们随后将确定它们对谷氨酸能突触和gaba能突触的最佳浓度和选择性。在目标3中，我们将执行一系列筛选后分析，以消除有毒化合物和假阳性。选择的化合物将被重新合成，结构确认，并重新测试以验证其突触标记。剩余的“高级命中”随后将被分类为“稳定”（在突触刺激期间荧光稳定），“比率”（在刺激期间发光特性改变）或“动态”（在胞外作用/突触活动期间化合物从突触中丢失）突触标记。最后，将使用实时成像方法评估它们对突触功能的影响。

DESCRIPTION (provided by applicant): Synapses are the essential components of neural circuits in the brain. It is widely accepted that a causal factor in many neuropsychiatric disorder is synaptic dysfunction, including abnormal synapse formation or defects in synaptic transmission. However, few tools are available for imaging synapse density and function in the living mammalian brain. Although viral delivery of genetically encoded probes (e.g. fluorescent proteins, channel rhodopsins) is widely used for live imaging/functional assays of synapses in the rodent brain, this approach is highly invasive, requires specialized techniques and reagents, and has had limited success in non-human primates, let alone humans. We therefore propose an alternative approach based upon the molecular recognition of native synaptic components by organic small molecules, thereby facilitating their selective localization to synapses. This "chemical" approach has the potential to extend the use of synaptic markers from rodent models into non-human primates and humans via non-invasive administration routes. Such synaptic markers could also serve as molecular targeting devices for the delivery of sensors and therapeutics to synapses in the living mammalian brain, affording transformative tools for neuroscience research, as well as for the diagnosis and treatment of neuropsychiatric disorders. As a first step toward these long-term goals, we propose in this application to develop a high-throughput screening (HTS) platform for discovery of small molecule fluorescent synaptic markers. We have obtained a library of ~8,000 novel fluorescent compounds based on diverse fluorophore structural cores, spanning a wide structural and spectroscopic range. This unique resource is a valuable tool for the discovery and development of synaptic markers. In Aim 1, we will develop a HTS assay using cultured cortical neurons in 96-well plate format. Synaptic labeling will be assessed based on colocalization with fluorescent protein (FP)-tagged synaptic vesicle-associated proteins that label presynaptic boutons. The screening, imaging, data mining, and hit selection protocols will be optimized with a preliminary screen of ~1,000-2,000 fluorescent compounds. In Aim 2, we will use protocols optimized in Aim 1 to screen the entire library of ~8,000 fluorescent dyes, and identify hit compounds that label synapses. We will subsequently determine their optimal concentrations and selectivity for glutamatergic versus GABAergic synapses. In Aim 3, we will perform a series of post-screening assays to eliminate toxic compounds and false positives. Select compounds will be resynthesized, structurally confirmed, and re-tested to validate their synaptic labeling. Remaining "advanced hits" will subsequently be classified as "stable" (fluorescence is stable during synaptic stimulation), "ratiometric" (light emission properties change during stimulation), or "dynamic" (compound is lost from synapses during exocytosis/synaptic activity) synaptic markers. Finally, their impact on synaptic function will be assessed using live imaging methods.