Abstracts

Rationale:
Loss-of-function mutations in NPRL3, a GATOR1 subunit and the key amino acid regulatory complex in the mTOR cascade, have been linked to malformations of cortical development (MCD) and epilepsy. Knockout (KO) of Nprl3 in vitro causes enhanced mTOR signaling and altered neuronal morphology. To further investigate the role of NPRL3 in brain development, MCD, and epilepsy, we hypothesized that CRISPR-editing of Nprl3 in vivo will cause altered cortical lamination, increased cell size, and seizures.
Method:
In utero electroporation was performed on E14 CD1 mouse embryos using Nprl3 CRISPR/Cas9 or scramble plasmids with a GFP reporter. Dams were injected with rapamycin (2.5 mg/kg) 24 hrs after surgery. Nprl3 CRISPR edits were validated by Nprl3 sequencing. Brains were removed from electroporated pups at P3 or at 5 weeks of age (n=5 per group) and processed for immunohistochemistry. Sections were probed with primary antibodies recognizing GFP (Cas9), Cux1, or MAP2 and then fluorescent secondary antibodies (alexa 488, 647, or 594, respectively). Brain section images were recorded on a spinning-disk confocal microscope and analyzed by binning images into cortical layers I, II, III, IV-VI, and white matter zones. Cell size measurements (n=20 per group) were taken from digital images using the longest aspect of the soma. Statistical analysis was performed using ANOVA. EEGs were implanted in 5 week old electroporated mice. Baseline EEG recordings were performed for 48 hrs followed by seizure threshold testing using PTZ challenge (n=5; 55 mg/kg). EEG analysis was performed using Sirenia Seizure Pro™. Latency to seizure (seconds), seizure severity (behavioral seizure score), and line length analysis were performed. Statistical analysis was performed using ANOVA.
Results:
P3 pups and adult mice electroporated with Nprl3 KO plasmid displayed GFP positive heterotopic neurons present in sub-cortical white matter vs. scramble electroporated neurons where GFP+ cells appeared only in layer II. P3 pups and adult mice also had increased soma diameter vs. scramble and WT controls (p< 0.05). Heterotopic localization and neuronal size changes were prevented in Nprl3 KO pups born from dams treated with rapamycin (n=5, p< 0.05). Adult Nprl3 KO mice implanted with EEGs did not have spontaneous seizures. However, Nprl3 KO mice displayed greater line length (a proxy for spike amplitude) in the electroporated cortex compared to the contralateral cortex (n=5). In addition, Nprl3 KO mice had decreased average latency to seizure (54.24 sec.) compared to WT control mice (78.29 sec.). Further, Nprl3 KO mice had increased PTZ-induced seizure severity based on behavioral seizure score (avg= 3; brief tonic-clonic seizure) compared to WT control mice (avg.= 1.75, abnormal posturing).
Conclusion:
Nprl3 KO results in cell size and lamination abnormalities in vivo which were prevented with rapamycin. Focal Nprl3 KO results in reduced seizure threshold and a hyperexcitable cortex in adult mice. These findings provide a model for human MCD and seizures associated with NPRL3 variants and suggests that mTOR inhibition could be considered in clinical trials.  
Funding:
:NIH RO1NS099452 and R01NS094596 (PBC)