Scalable, all-optical assays of synaptic function and plasticity

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。 尽管这些疾病的患病率和严重性，但新疗法的开发已经滞后。这 部分是由于在稳健的、可扩展的体外测定中复制相关生物学的挑战。当前方法 测量突触功能，刺激突触前细胞和记录从突触后细胞，缺乏 用于药物筛选的足够通量。最近在Q-State Biosciences开发的Optopatch平台， 由工程光遗传学蛋白，定制显微镜和软件组成，使得有可能 在1毫秒内同时刺激（蓝光）和记录（红光）约100个神经元电活动 时间分辨率、单细胞空间分辨率和高信噪比。此外，图案化的蓝光 可以通过刺激单个神经元来探测突触连接，同时记录突触后 在所有剩余的细胞中的电位（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，用于： 1.突触前钙-红色钙敏感蛋白jRGECO 1a通过以下方式靶向突触前终扣： 与突触素融合。神经活动是通过一种通道视紫红质CheRiff用蓝光刺激的。 2.突触后钙-jRGECO 1a通过与PSD 95融合靶向突触后棘。不同 神经元的子集表达致动器或报告子。突触前细胞触发的动作电位 在突触后细胞中产生钙信号。 3.突触后电压- CheRiff和红色电压传感蛋白QuasAr在不同的细胞中表达。 神经元的子集。突触前细胞刺激导致在QuasAr表达中记录的PSP 突触后细胞。药理学探针通过AMPA或NMDA分离兴奋性信号传导 通道或通过GABAA通道的抑制性信号。抑制性神经元可以用 在Dlx 1/2启动子的控制下表达的荧光标签，以分辨不同的突触类别： 兴奋性（E）→抑制性（I）、E →E、I →E和I → I。 在后续的II期项目中，我们建议：（1）将检测方法过渡到人诱导多能干细胞 衍生的神经元，测试多种策略以增加细胞的突触成熟，（2）扩展测定 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， 和（3）在ASD疾病模型中使用敲除三种突触蛋白，SHANK 3， SYNGAP 1和GRIN 2B，它们的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）筛选已批准药物的库，以证明测定通量和灵敏度，并鉴定候选药物 可能的再利用建立HTS的ASD相关细胞表型将提供一种新的治疗方法。 为这些严重和治疗不善的疾病的药物发现奠定了基础。

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.