mTOR Activation Causes Cellular Aggregation and Proteomic Changes in vitro
Abstract number :
3.027
Submission category :
1. Basic Mechanisms / 1B. Epileptogenesis of genetic epilepsies
Year :
2025
Submission ID :
1175
Source :
www.aesnet.org
Presentation date :
12/8/2025 12:00:00 AM
Published date :
Authors :
Kelley Roark, BS – University of Maryland School of Medicine
Presenting Author: Peter Crino, MD, PhD – University of Maryland School of Medicine
Philip Iffland, PhD – University of Maryland Baltimore
Rationale: Malformations of cortical development (MCDs) associated with epilepsy result from germline or somatic mutations in mTOR pathway genes (‘mTORopathies’). mTORopathies such as focal cortical dysplasia type 2 (FCD2) are defined histologically by cytomegalic dysmorphic neurons, balloon cells, and heterotopic neurons in white matter. Neurons in MCD are frequently found in close apposition to each other and aggregates of cells are often observed in experimental mTORopathy models. We hypothesized that CRISPR/Cas9 knockout (KO) of mTOR pathway genes (MPG) Tsc2, Nprl3, Strada, or Kptn in N2a cells in vitro would result in mTOR-dependent cellular aggregation and downstream changes in adhesion molecule expression.
Methods: CRISPR/Cas9 KO of N2a cell (N2aC) lines were generated for MPG (Tsc2, Nprl3, Kptn, and Strada) along with scramble gRNAs, and wildtype N2aC controls. N2aC for each line were cultured in video dishes for live cell imaging and chamber slides for immunocytochemistry. MPG KO was confirmed by western blot or RT-PCR. Treatment groups included mTOR inhibitors rapamycin (50 nM; 48hrs) or torin1 (50 nM; 48 hrs), or physiological media only. All cells were plated at equal density. Video dishes were maintained in an imaging incubator and images were taken every 15 minutes for 48 hours. Images were compiled for video analysis. Chamber slide samples were fixed in paraformaldehyde and probed with F-actin antibodies and fluorescent secondary antibodies. Aggregate volume, density, and number were measured using digital micrographs and ANOVA statistical analysis was performed (p< 0.05 deemed significant). Protein lysates from cellular aggregates were collected and sent for mass spectrometry analysis.
Basic Mechanisms