Authors :
Presenting Author: Kelley Roark, BS – University of Maryland School of Medicine
Aditi Biswas, BS, MPH – University of Maryland School of Medicine
Sophie Bruckmier, PhD – University of Maryland School of Medicine
Seth Ament, PhD – University of Maryland School of Medicine
Philip Iffland, PhD – University of Maryland Baltimore
Rationale:
Focal Cortical Dysplasia (FCD) is a leading cause of drug-resistant epilepsy and is often associated with somatic mutations in mTOR pathway repressors such as NPRL2. While mTORC1 inhibition with rapamycin has been shown to partially rescue structural abnormalities in experimental models and a reduction in seizure frequency in patients, emerging evidence suggests that bioenergetic and metabolic immaturity may persist. Spatial Transcriptomic analysis of cortical layers II/III, where Nprl2 KO neurons are concentrated following in utero electroporation, revealed sustained dysregulation of key metabolic genes (e.g., Fndc5, Hs6st2, Dok5) despite rapamycin treatment. Therefore, we hypothesized that mTORC1 inhibition alone may be insufficient to restore mitochondrial function in vitro.
Methods:
CRISPR/Cas9-generated Nprl2 knockout Neuro2a (N2a), scramble control, and WT N2a cells were used were used to assay mitochondrial respiration and glycolytic function using Seahorse XF extracellular flux analysis. Treatment conditions were rapamycin (50 nM, 48 hrs) and dichloroacetate (DCA; 1 mM, 48 hrs), a metabolic modulator that enhances oxidative metabolism by facilitating pyruvate entry into the TCA cycle. Redox status was measured via NAD⁺/NADH assays, and transcriptional responses were monitored by qPCR. Group comparisons were evaluated using one-way ANOVA with p < 0.05 considered significant. Experiments were performed with n = 5 biological replicates per condition.
Results:
Our findings indicate altered oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and NAD⁺/NADH ratios in Nprl2 KO cells relative to controls, suggesting the cells remain metabolically immature and locked in a glycolytic state. Treatment with rapamycin and DCA was evaluated for potential rescue of metabolic function and resulted in modest improvements in OCR and redox balance. Differential expression of metabolic genes in KO vs KO+RAP conditions was used to guide interpretation of mitochondrial phenotype and therapeutic response and transcriptomic trends were consistent with persistent metabolic immaturity.
Conclusions:
We observed that Nprl2 KO N2a cells retain a persistent glycolytic phenotype that is only partially rescued by rapamycin or DCA treatment. These data suggest that neuronal metabolic immaturity is a sustained and targetable feature of mTOR-opathy-associated epilepsy, with implications for future therapeutic strategies aimed at restoring mitochondrial function in FCD. Transcriptomic evidence of persistent metabolic gene dysregulation supports this, and the need for bioenergetic profiling in mTORopathy models and may inform future therapeutic strategies for FCD-associated epilepsy.
Funding:
NIH NINDS RO1NS131223 (PHI) and NINDS diversity supplement to RO1NS131223 (KMR).