High-throughput screening platform for discovery of fluorescent synaptic markers

用于发现荧光突触标记的高通量筛选平台

基本信息

批准号：
8769206
负责人：
Clarissa Leigh Waites
金额：
$ 24万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-15 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8769206
关键词：
Aging Autistic Disorder Biological Assay Brain Calcium Chemicals Clinical Communication Data Defect Development Devices Diagnosis Disease Dyes Exocytosis Fluorescence Fluorescent Dyes Functional Imaging Functional disorder Glutamates Goals Human Image Individual Label Learning Libraries Life Major Depressive Disorder Molecular Biology Molecular Target Monitor Neurodevelopmental Disorder Neurons Neurosciences Neurosciences Research Organic Synthesis Physiology Poison Process Property Proteins Protocols documentation Reagent Resources Rhodopsin Rodent Rodent Model Route Schizophrenia Scientist Series Signal Transduction Site Synapses Synaptic Transmission Synaptic Vesicles Synaptic plasticity Techniques Testing Therapeutic Time Toxic effect Viral base calcium indicator data mining density design fluorophore follow-up high throughput screening imaging modality imaging probe light emission molecular recognition neural circuit neuronal survival neuropsychiatry nonhuman primate novel novel strategies optical imaging presynaptic public health relevance ratiometric screening sensor small molecule small molecule libraries success synaptic function synaptogenesis tool voltage

项目摘要

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.

描述（由申请人提供）：突触是大脑神经回路的重要组成部分。人们普遍认为，许多神经精神疾病的致病因素是突触功能障碍，包括突触形成异常或突触传递缺陷。然而，很少有工具可用于对活体哺乳动物大脑中的突触密度和功能进行成像。尽管基因编码探针（例如荧光蛋白、通道视紫红质）的病毒递送广泛用于啮齿动物大脑中突触的实时成像/功能测定，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，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，从而促进它们选择性定位到突触。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，将传感器和治疗药物传递到活体哺乳动物大脑中的突触，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，我们在本申请中建议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。我们已经获得了约 8,000 种基于不同荧光团结构核心的新型荧光化合物库，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的宝贵工具。在目标 1 中，我们将使用 96 孔板形式培养的皮质神经元开发 HTS 测定。突触标记将基于与荧光蛋白 (FP) 标记的突触小泡相关蛋白的共定位来评估，这些蛋白标记了突触前的连接。筛选、成像、数据挖掘和命中选择方案将通过约 1,000-2,000 种荧光化合物的初步筛选进行优化。在目标 2 中，我们将使用目标 1 中优化的方案来筛选约 8,000 种荧光染料的整个库，并识别标记突触的命中化合物。我们随后将确定它们对谷氨酸能突触与 GABA 能突触的最佳浓度和选择性。在目标 3 中，我们将进行一系列筛选后检测，以消除有毒化合物和误报。选定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”（荧光在突触刺激期间稳定）、“比例”（刺激期间光发射特性改变）或“动态”（在胞吐作用/突触活动期间化合物从突触丢失）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。