Abstracts

Rationale: Malformations of cortical development (MCD) account for an estimated 40% of childhood refractory epilepsies. The most common cause of MCD are germline and somatic variants in mTOR pathway genes ("mTORopathies"), which is defined by mTOR pathway hyperactivation and dysmorphic neurons. Variants in subunits of the GATOR1 complex (DEPDC5, NPRL3 and NPRL2) are the most common cause of focal cortical dysplasia (FCD). It is known that Depdc5 and Nprl3 knockout (KO) result in mTOR pathway hyperactivation, increased cell size, laminar defects, and seizures in model systems. While NPRL2 performs the primary GTPase function of GATOR1, few studies have attempted to functionally validate or model FCD-associated variants in NPRL2.

Methods: CRISPR/Cas9 was used to create an Nprl2 KO cell line in Neuro 2a cells (N2aC). KO was validated using qPCR and Western assay. WT, scramble (SC), and KO N2aC were treated with mTOR inhibitors (rapamycin and torin1, 150 nM) in complete media or treated with amino acid free media to assess phosphorylated ribosomal S6 levels (PS6 240/244). Cell lines were treated with vehicle or mTOR inhibitors and visualized via immunofluorescence staining with F-actin to measure cell soma size and assayed by imaging cytometry. Time-lapse imaging was used to capture aggregation patterns in all 3 cell lines with and without mTOR inhibitors, and morphology was visualized via F-actin probing and spinning disk confocal microscopy. CD1 mouse embryos underwent in utero electroporation (IUE) using Nprl2 CRISPR/Cas9 or scramble plasmids with a GFP reporter at E14. Dams were injected with rapamycin (2.5 mg/kg) 24 hrs after surgery. At P3 or at 5 weeks of age, brains were dissected from IUE pups and processed for immunohistochemistry. Sections were probed with primary antibodies recognizing GFP or CTIP2 and then fluorescent secondary antibodies (alexa 488, 647). Brain sections were imaged on a Keyence microscope and analyzed by binning images into cortical layers I, II, III, IV-VI, and white matter zones. Finally, the longest aspect of the soma was used to measure cell size in tissue slices

Results: Nprl2 KO showed significantly reduced mRNA expression by qPCR and significantly reduced protein expression by WB. Nprl2 KO resulted in mTOR-dependent increases in pS6, which was reversible with rapamycin treatment. KO cells also showed increased soma size vs WT cells, which was reduced with rapamycin treatment. KO cells formed abnormal aggregates that were larger in size, volume, and cell number than control cells. Aggregate formation was preventable with mTOR inhibitors. Nprl2 IUE mice displayed GFP+ heterotopic neurons in sub-cortical white matter while SC IUE  neurons displayed GFP+ cells only in layer II. Nprl2 IUE mice also had increased soma diameter vs SC and WT controls. These changes were prevented in Nprl2 KO pups born from rapamycin treated dams.

Conclusions: Nprl2 KO results in mTOR pathway hyperactivation and altered morphology in vitro, and heterotopic neurons in vivo -consistent with FCD phenotypes. These finding provide pre-clinical support for the use of mTOR inhibitors in individuals with Nprl2 associated FCD.

Funding: American Epilepsy Society Junior Investigator Award to PHI