Scalable, all-optical assays of synaptic function and plasticity

项目摘要：突触功能障碍与包括癫痫在内的许多神经系统疾病有关。 阿尔茨海默氏症、帕金森氏症、自闭症谱系障碍(ASD)、精神分裂症、抑郁症、多动症和亨廷顿氏症。 尽管这些疾病的流行率和严重性都很高，但新疗法的开发一直滞后。这 这在一定程度上是由于在稳健、可扩展的体外试验中复制相关生物学的挑战。当前的方法 测量突触功能，刺激突触前细胞并记录突触后细胞，缺乏 足以进行药物筛选的吞吐量。Q-State生物科学公司最近开发的Optopatch平台， 由工程光遗传蛋白、定制显微镜和软件组成，使其有可能 在1毫秒内同时刺激(蓝光)和记录(红光)~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，图案化蓝光 可用于通过在记录突触后刺激单个神经元来探测突触连接 所有剩余细胞的电位(PSP)。在第一阶段，我们在原代啮齿动物神经元中开发了突触分析，用于： 1.突触前钙--红色钙感应蛋白jRGECO1a通过 与突触素融合。神经活动是由蓝光通过视紫红质通道刺激的，切里夫。 2.通过与PSD95融合，突触后钙-jRGECO1a被靶向突触后棘。截然不同 神经元的亚群表达执行器或报告器。突触前细胞中触发的动作电位 在突触后细胞中产生钙信号。 3.突触后电压-切里夫和红色电压感应蛋白类星体在 神经元的子集。突触前细胞刺激导致类星体表达的PSP 突触后细胞。药理探针通过AMPA或NMDA分离兴奋性信号 通道或通过GABAA通道的抑制信号。抑制性神经元可以用一种 在Dlx1/2启动子控制下表达的荧光标记，以解析不同的突触类型： 兴奋性(E)→抑制(I)、E→E、I→E和I→I。 在后续的第二阶段项目中，我们建议：(1)将检测过渡到人诱导的多能干细胞 衍生神经元，测试增加细胞突触成熟的多种策略，(2)扩展分析 在啮齿动物细胞中包括可塑性，特别是长时程增强(LTP)和依赖于峰时的可塑性， 以及(3)通过敲除三种突触蛋白SHANK3，将这些检测方法应用于ASD疾病模型。 Syngap1和GRIN2B，它们的缺失在所有情况下都会导致严重的ASD。将使用最健壮的表型 (4)筛选已批准药物的库，以证明检测吞吐量和敏感性，并确定候选药物 用于潜在的再利用。ASD相关细胞表型的建立将为HTS提供一种 为治疗这些严重和治疗不善的疾病而建立的药物发现基金会。

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.