Abstracts

Rationale: The most common type of drug-resistant epilepsy in children results from focal cortical dysplasia (FCD type 2; FCD2), a type of cortical malformation. FCD2 is 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 hypothesize that Nprl2 KO will result in mTOR-dependent changes in neuronal morphology in vivo.

Methods: CD1 mouse embryos underwent in utero electroporation (IUE) using Nprl2 CRISPR/Cas9 or scramble plasmids at E14. Dams were injected with rapamycin, torin1, rapalink, or an mTORC2 inhibitor 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 on a fluorescent microscope and analyzed by binning images into cortical layers I,-III, IV-VI, and white matter zones. Digital micrographs were analyzed for changes in neuronal morphology, mTOR signaling, and neuronal migration. A separate cohort of animals was matured to P30 and implanted with dural EEG electrodes. EEGs were recorded for 5 days and seizure threshold testing, using PTZ, was performed. EEGs were analyzed for spontaneous seizures and other interictal abnormalities, as well as changes in EEG power and latency to seizure after seizure threshold testing.

Results: P3 mouse pups electroporated with Nprl2 KO plasmid 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 electroporated neurons where GFP+ cells appeared only in upper cortical layers. In Nprl2 KO pups, migratory defects were rescued with rapamycin, torin1, or rapalink treatment but not mTORC2 inhibitor treatment (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 mouse brain specimens that were rescued with mTORC1 but not mTORC2 inhibition (p< 0.05). Lastly, we did not observe spontaneous behavioral or electrographic seizures in Nprl2 KO animals. However, a significant reduction in seizure threshold, as measured by latency to first seizure, was observed in Nprl2 KO animals relative to control. Latency to seizure was rescued with mTORC1 but not mTORC2 inhibition.