Departments of Neurosurgery, and Cellular and Molecular Physiology, Wu Tsai Institute, Yale University School of Medicine, New Haven, CT 06520-8082.
Departments of Neurosurgery, and Cellular and Molecular Physiology, Wu Tsai Institute, Yale University School of Medicine, New Haven, CT 06520-8082
eNeuro. 2023 Aug 28;10(8). doi: 10.1523/ENEURO.0160-23.2023. Print 2023 Aug.
Several neurodevelopmental disorders are associated with increased mTOR activity that results in pathogenic neuronal dysmorphogenesis (i.e., soma and dendrite overgrowth), leading to circuit alterations associated with epilepsy and neurologic disabilities. Although an mTOR analog is approved for the treatment of epilepsy in one of these disorders, it has limited efficacy and is associated with a wide range of side effects. There is a need to develop novel agents for the treatment of mTOR-pathway related disorders. Here, we developed a medium-throughput phenotypic assay to test drug efficacy on neurite morphogenesis of mouse neurons in a hyperactive mTOR condition. Our assay involved electroporation (IUE) of a selective population of cortical pyramidal neurons with a plasmid encoding the constitutively active mTOR activator, Rheb, and tdTomato. Labeled neurons from the somatosensory cortex (SSC) were cultured onto 96-well plates and fixed at various days or following Torin 1 treatment. Automated systems were used for image acquisition and neuron morphologic measurements. We validated our automated approach using traditional manual methods of neuron morphologic assessment. Both automated and manual analyses showed increased neurite length and complexity over time, and decreased neurite overgrowth and soma size with Torin 1. These data validate the accuracy of our automated approach that takes hours compared with weeks when using traditional manual methods. Taken together, this assay can be scaled to screen 32 compounds simultaneously in two weeks, highlighting its robustness and efficiency for medium-throughput screening of candidate therapeutics on a defined population of wild-type or diseased neurons.
几种神经发育障碍与 mTOR 活性增加有关，导致病理性神经元发育不良（即体和树突过度生长），导致与癫痫和神经功能障碍相关的回路改变。虽然一种 mTOR 类似物已被批准用于治疗其中一种疾病的癫痫，但它的疗效有限，且与广泛的副作用有关。因此，需要开发新的药物来治疗 mTOR 通路相关疾病。在这里，我们开发了一种高通量表型测定法，用于测试在 mTOR 过度活跃的情况下，药物对小鼠神经元神经突形态发生的疗效。我们的测定法涉及用电穿孔（IUE）将编码组成型激活的 mTOR 激活剂 Rheb 和 tdTomato 的质粒转染到选择性的皮质锥体神经元群体中。来自体感皮层（SSC）的标记神经元被培养在 96 孔板上，并在不同的时间点或在 Torin 1 处理后固定。自动系统用于图像采集和神经元形态测量。我们使用传统的神经元形态评估手动方法验证了我们的自动化方法的准确性。自动和手动分析都显示出随着时间的推移神经突长度和复杂性增加，而 Torin 1 则减少了神经突过度生长和体大小。这些数据验证了我们的自动化方法的准确性，与使用传统手动方法相比，该方法需要数小时，而不是数周。总之，该测定法可以在两周内同时筛选 32 种化合物，突出了其在针对特定野生型或疾病神经元群体的候选治疗药物进行高通量筛选中的稳健性和效率。
