Scalable, all-optical assays of synaptic function and plasticity

突触功能和可塑性的可扩展、全光学测定

• 批准号: 9916820
• 负责人: Graham Thomas Dempsey
• 金额: $ 77.12万
• 依托单位: Q-STATE BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-12 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9916820
• 关键词: Action Potentials; Address; Alzheimer' s Disease; Attention deficit hyperactivity disorder; Biological Assay; Biological Models; Biological Sciences; Biology; CRISPR/Cas technology; Calcium; Calcium Signaling; Cell Maturation; Cells; Chemicals; Coculture Techniques; Collaborations; Complex; Computer software; Coupled; Custom; DLG4 gene; Data Set; Depressed mood; Development; Disease; Disease model; Drug Screening; Electrophysiology (science); Engineering; Environment; Epilepsy; Exhibits; Fire - disasters; Foundations; Frequencies; Functional disorder; Genes; Genetic; Grant; Growth Factor; Health; Human; Huntington Disease; Image; Individual; Industrialization; Ions; Kinetics; Knock-out; Label; Libraries; Light; Link; Long-Term Potentiation; Measurement; Measures; Mediating; Mental Depression; Mental disorders; Methods; Microscope; Modeling; Mus; N-Methylaspartate; Neurodevelopmental Disorder; Neurologic; Neuromodulator; Neurons; Noise; Optics; Parkinson Disease; Patients; Pattern; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phase; Phenotype; Physiological; Positioning Attribute; Prevalence; Proteins; Protocols documentation; Reporter; Resolution; Rodent; Schizophrenia; Severities; Signal Transduction; Small Business Innovation Research Grant; Synapses; Synaptic Transmission; Synaptic plasticity; Synaptophysin; Testing; Therapeutic; Validation; Vertebral column; Work; autism spectrum disorder; base; commercialization; disease phenotype; disease-causing mutation; drug discovery; excitatory neuron; functional status; gamma-Aminobutyric Acid; high throughput screening; improved; in vitro Assay; in vitro Model; induced pluripotent stem cell; inhibitory neuron; interest; loss of function mutation; millisecond; mouse model; nervous system disorder; neuropsychiatric disorder; novel drug class; novel therapeutics; optogenetics; postsynaptic; presynaptic; programs; promoter; quasar; relating to nervous system; response; screening; synaptic function; temporal measurement; therapeutic candidate; voltage

Project Summary: Synaptic dysfunction has been implicated in many neurological diseases including epilepsy, Alzheimer’s, Parkinson’s, autism spectrum disorder (ASD), schizophrenia, depression, ADHD and Huntington’s. Despite the prevalence and severity of these disorders, the development of new therapeutics has lagged. This is due, in part, to challenges in replicating relevant biology in robust, scalable in vitro assays. Current methods of measuring synaptic function, which stimulate presynaptic cells and record from postsynaptic cells, lack sufficient throughput for drug screening. The Optopatch platform recently developed at Q-State Biosciences, comprised of engineered optogenetic proteins, custom microscopes, and software, makes it possible to simultaneously stimulate (blue light) and record (red light) electrical activity from ~100 neurons with 1 millisecond temporal resolution, single-cell spatial resolution and high signal-to-noise ratio. Additionally, patterned blue light can be used to probe synaptic connections by stimulating individual neurons while recording postsynaptic potentials (PSPs) in all remaining cells. In Phase I, we developed synaptic assays in primary rodent neurons for: 1. Presynaptic calcium – The red calcium sensing protein jRGECO1a is targeted to presynaptic boutons by fusion with synaptophysin. Neural activity is stimulated with blue light via a channelrhodopsin, CheRiff. 2. Postsynaptic calcium – jRGECO1a is targeted to postsynaptic spines by fusion with PSD95. Distinct subsets of neurons express either actuator or reporter. Action potentials triggered in presynaptic cells generate calcium signals in postsynaptic cells. 3. Postsynaptic voltage – CheRiff and the red voltage sensing protein QuasAr are expressed in distinct subsets of neurons. Presynaptic cell stimulation leads to PSPs recorded in QuasAr-expressing postsynaptic cells. Pharmacological probes isolate excitatory signaling through either AMPA or NMDA channels or inhibitory signaling through GABAA channels. Inhibitory neurons can be labeled with a fluorescent tag expressed under control of the Dlx1/2 promoter, to resolve different synapse classes: excitatory (E) → inhibitory (I), E →E, I →E, and I → I. In the follow-on Phase II project, we propose to: (1) transition the assays to human induced pluripotent stem cell derived neurons, testing multiple strategies to increase the synaptic maturation of the cells, (2) expand assays in rodent cells to include plasticity, particularly long-term potentiation (LTP) and spike timing dependent plasticity, and (3) apply these assays in disease models of ASD using knockout of three synaptic proteins, SHANK3, SYNGAP1, and GRIN2B, whose loss causes severe ASD in all cases. The most robust phenotype will be used to (4) screen a library of approved drugs to demonstrate assay throughput and sensitivity and identify candidates for potential repurposing. The establishment of ASD-associated cellular phenotypes for HTS would provide a foundation for drug discovery for these serious and poorly treated diseases.

项目摘要：突触功能障碍与许多神经系统疾病有关，包括癫痫、 阿尔茨海默病、帕金森病、自闭症谱系障碍 (ASD)、精神分裂症、抑郁症、多动症和亨廷顿舞蹈症。 尽管这些疾病普遍存在且严重，但新疗法的开发仍然滞后。这 部分原因是在稳健、可扩展的体外测定中复制相关生物学的挑战。目前的方法 测量突触功能（刺激突触前细胞并记录突触后细胞）的方法缺乏 足够的通量用于药物筛选。 Q-State Biosciences 最近开发了 Optopatch 平台， 由工程光遗传学蛋白质、定制显微镜和软件组成，使得 在 1 毫秒内同时刺激（蓝光）并记录（红光）约 100 个神经元的电活动 时间分辨率、单细胞空间分辨率和高信噪比。此外，图案蓝光 可用于通过刺激单个神经元同时记录突触后来探测突触连接 所有剩余细胞中的电位（PSP）。在第一阶段，我们在初级啮齿动物神经元中开发了突触测定法，用于： 1. 突触前钙 – 红色钙传感蛋白 jRGECO1a 通过以下方式靶向突触前 boutons 与突触素融合。蓝光通过视紫红质 CheRiff 刺激神经活动。 2. 突触后钙 – jRGECO1a 通过与 PSD95 融合而靶向突触后棘。清楚的 神经元子集表达执行器或报告器。突触前细胞触发动作电位 在突触后细胞中产生钙信号。 3. 突触后电压——CheRiff 和红色电压传感蛋白 QuasAr 以不同的形式表达 神经元的子集。突触前细胞刺激导致记录在 QuasAr 表达中的 PSP 突触后细胞。药理学探针通过 AMPA 或 NMDA 分离兴奋信号 通道或通过 GABAA 通道的抑制信号传导。抑制性神经元可以用 在 Dlx1/2 启动子控制下表达的荧光标签，用于解析不同的突触类别： 兴奋性 (E) → 抑制性 (I)，E →E，I →E，I → I。 在后续的二期项目中，我们建议：（1）将检测方法转向人诱导多能干细胞 衍生神经元，测试多种策略以促进细胞突触成熟，(2) 扩大测定 在啮齿动物细胞中包括可塑性，特别是长时程增强（LTP）和尖峰时间依赖性可塑性， (3) 通过敲除三种突触蛋白 SHANK3，将这些测定法应用于自闭症谱系障碍 (ASD) 疾病模型中 SYNGAP1 和 GRIN2B，其缺失在所有情况下都会导致严重的 ASD。将使用最稳健的表型 (4) 筛选已批准药物库以证明检测通量和灵敏度并识别候选药物 用于潜在的重新利用。 HTS 的 ASD 相关细胞表型的建立将为 为治疗这些严重且治疗不佳的疾病的药物发现奠定了基础。