Spatial transcriptomics reveal mTOR-dependent and mTOR-independent changes in cortical development in a model of focal cortical dysplasia
Abstract number :
1.018
Submission category :
1. Basic Mechanisms / 1B. Epileptogenesis of genetic epilepsies
Year :
2025
Submission ID :
1129
Source :
www.aesnet.org
Presentation date :
12/6/2025 12:00:00 AM
Published date :
Authors :
Aditi Biswas, BS, MPH – University of Maryland School of Medicine
Sophie Bruckmeier, Ph.D – University of Maryland School of Medicine
David Kolb, BS – University of Maryland School of Medicine
Adrian Auber, BS – St. Mary's College of Maryland
Seth Ament, PhD – University of Maryland School of Medicine
Presenting Author: Philip Iffland, PhD – University of Maryland Baltimore
Rationale: Focal Cortical Dysplasia Type 2 (FCD2) commonly caused by variants in genes that encode proteins forming the GATOR1 complex (NPRL3, DEPDC5, and NPRL2)- a negative regulator of the mTOR pathway. FCD2 phenotypes such as abnormal neuronal morphology and heterotopic neurons are linked to mTOR hyperactivation. While NPRL3 and DEPDC5 variants are well defined, NPRL2 variants are emerging as another frequent cause of FCD2. We hypothesized that Nprl2 KO would result in mTOR-dependent and mTOR-independent changes within in the developing cerebral cortex that would explain common FCD2 phenotypes.
Methods: CD1 mouse embryos underwent in utero electroporation (IUE) using Nprl2 CRISPR/Cas9 or scramble plasmids at E14. Dams were injected with rapamycin 24 hrs after surgery. At P3 and P40, brains were dissected from animals and processed by immunochemistry probing for GFP, SATB2 (layer II/III), and CTIP2 (layer IV-VI). Brain sections were imaged and analyzed by binning cortical layers I-III, IV-VI, and white matter. Digital micrographs were analyzed for changes in neuronal morphology, mTOR signaling, and neuronal migration. Another cohort of animals was matured to P30 and implanted with EEG electrodes. EEGs were recorded for 5 days and seizure threshold testing was performed. EEGs were analyzed for spontaneous seizures or interictal abnormalities, as well as changes in EEG power and latency to seizure after seizure threshold testing. Curio Seeker (Slide-seq) spatial transcriptomics was used to produce cortical atlases of neonatal mice in three groups: Nprl2 KO, Nprl2 KO with rapamycin, and controls.
Results: Nprl2 KO pups displayed migratory defects and increased cell size (n= 50; p< 0.05) as evidenced by GFP and CUX1 positive neurons dispersed throughout the electroporated region vs. scramble control. Migratory defects were rescued with rapamycin (n=6 animals per group; p< 0.05). mTOR-dependent increases in soma diameter and increased mTOR pathway signaling were observed in P3 and P40 brain specimens (p< 0.05). We did not observe spontaneous seizures in Nprl2 KO animals. However, a significant reduction in seizure threshold was observed in Nprl2 KO animals vs. control. Nprl2 KO was associated with 201 cortical layer-specific gene expression changes (FDR < 0.05), including layer-specific dysregulation of neurodevelopmental transcription factors and synaptic components, as well as broader activation of neuroinflammatory signatures. Laminar identity in layer 2/3 was preserved across groups, but layer 5 markers were ectopically expressed in superficial layers in
Basic Mechanisms