Abstracts

Rationale: One emerging cause of focal epilepsy is mutations in the gene nitrogen permease regulator-like 2 (NPRL2). NPRL2 is a part of the gap activity toward RAGS 1 (GATOR1) complex and functions to negatively regulate the mechanistic target of rapamycin complex 1 (mTORC1). The majority of patients with GATOR1 mutations have treatment resistant epilepsy and are thus at greater risk of SUDEP. However, mechanisms underlying this susceptibility to epilepsy remains poorly understood.

Methods: To investigate mechanisms underlying seizures with NPRL2 loss and because of lethality associated with constitutive mutants we generated conditional Nprl2 mutants. We generated two mouse models of conditional neuronal Nprl2 loss: a pan-neuronal and glial model (Nestin^cre) and an excitatory neuronal conditional mutant with deletion in the forebrain and hippocampus (Emx1^cre). Both mutants displayed seizure activity and early lethality. To investigate potential mechanisms driving epilepsy, we utilized the Emx1^cre mutants. Neocortical tissue was isolated to perform LC/MS metabolomics studies, n=6 mice for each group, studies which revealed glycine as a potential contributory mechanism. To investigate this possibility, acute slice recordings were performed on mice age postnatal day (P)15-20 in the setting of strychnine, 7-Cl-Kynuerenic Acid, CPP to evaluate the contribution of specific glycine receptors to synaptic changes. Probenecid treatments started at p12, given daily TID. 

Results: Homozygous mutant Nprl2 mice with the Nestin^cre promoter exhibited early mortality, dying by P8-12 (n=8 control, 4 mutant, p< 0.0001). Mutant mice weighed much less than heterozygote and control littermates (n= 3 control, 2 het, 2 mutant, p= 0.009 and < 0.0001 respectively) and demonstrated spontaneous seizures. The Emx1^cre homozygous mutant mice for Nprl2 also died young and displayed spontaneous seizures, but did so only by P21. They too weighed less than heterozygote and control littermates. Heterozygotes in both lines did not exhibit spontaneous seizures, nor did they have early mortality. Because of the increased survival, the Emx1^cre model was selected for initial mechanistic studies. Physiologic studies revealed evidence of both increased excitatory and decreased inhibitory synaptic changes (Figure 1A-F) while metabolic studies revealed an increase in the amino acid and neurotransmitter glycine (Figure 2A). Further acute slice recordings support that blocking the co-agonist glycine_B site at the NMDAR ameliorated the increased excitatory synaptic changes present in the mutant (Figure 2B-E). Probenecid treatments to inhibit glycine’s actions at the NMDAR does increase survival in mutants (Figure 2F-G).

Conclusions: Brain specific deletion of Nprl2 results in spontaneous seizures in addition to synaptic and metabolic disruptions.  We identify glycine actions on the NMDAR as a significant contributor to synaptic changes and survival. These findings are significant because further examination of this mechanism to seizure phenotype will be an important avenue of future study.

Funding: Benjamin P. Tu acknowledges CPRIT RP140655 and R01 NS115546. Peter T. Tsai acknowledges DOD W81XWH-17-1-0238 and institutional startup funds.