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In Utero Electroporated Neurons for Medium-Throughput Screening of Compounds Regulating Neuron Morphology
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...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Society for Neuroscience
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10464655/ https://www.ncbi.nlm.nih.gov/pubmed/37620147 http://dx.doi.org/10.1523/ENEURO.0160-23.2023 |
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author | Sokolov, Aidan M. Aurich, Mariana Bordey, Angélique |
author_facet | Sokolov, Aidan M. Aurich, Mariana Bordey, Angélique |
author_sort | Sokolov, Aidan M. |
collection | PubMed |
description | 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 in utero 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 in vitro 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. |
format | Online Article Text |
id | pubmed-10464655 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Society for Neuroscience |
record_format | MEDLINE/PubMed |
spelling | pubmed-104646552023-08-30 In Utero Electroporated Neurons for Medium-Throughput Screening of Compounds Regulating Neuron Morphology Sokolov, Aidan M. Aurich, Mariana Bordey, Angélique eNeuro Research Article: Methods/New Tools 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 in utero 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 in vitro 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. Society for Neuroscience 2023-08-28 /pmc/articles/PMC10464655/ /pubmed/37620147 http://dx.doi.org/10.1523/ENEURO.0160-23.2023 Text en Copyright © 2023 Sokolov et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. |
spellingShingle | Research Article: Methods/New Tools Sokolov, Aidan M. Aurich, Mariana Bordey, Angélique In Utero Electroporated Neurons for Medium-Throughput Screening of Compounds Regulating Neuron Morphology |
title | In Utero Electroporated Neurons for Medium-Throughput Screening of Compounds Regulating Neuron Morphology |
title_full | In Utero Electroporated Neurons for Medium-Throughput Screening of Compounds Regulating Neuron Morphology |
title_fullStr | In Utero Electroporated Neurons for Medium-Throughput Screening of Compounds Regulating Neuron Morphology |
title_full_unstemmed | In Utero Electroporated Neurons for Medium-Throughput Screening of Compounds Regulating Neuron Morphology |
title_short | In Utero Electroporated Neurons for Medium-Throughput Screening of Compounds Regulating Neuron Morphology |
title_sort | in utero electroporated neurons for medium-throughput screening of compounds regulating neuron morphology |
topic | Research Article: Methods/New Tools |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10464655/ https://www.ncbi.nlm.nih.gov/pubmed/37620147 http://dx.doi.org/10.1523/ENEURO.0160-23.2023 |
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