In utero electroporated neurons for medium-throughput screening of compounds regulating neuron morphology

Abstract

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 neurological 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. Herein, 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 in utero electroporation 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 were cultured onto 96-well plates and fixed at various days in vitro or following Torin 1 treatment. Automated systems were utilized for image acquisition and neuron morphological measurements. We validated our automated approach using traditional manual methods of neuron morphological 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 to 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 wildtype or diseased neurons.

Significance Statement

Preclinical studies and screens often rely on cell lines and traditional techniques that are time consuming and introduce human bias during analysis compared to automated methods. Some of these techniques include the manual tracing of cells to quantify morphological changes in response to various treatments. We developed an assay that allows medium-throughput morphological analysis in a selective population of neurons in vitro while expediting data collection and analysis through automation. This assay is modifiable and is applicable for a wide range of disease conditions.