De novo mutations in the GRIN2A gene, which encodes the GluN2A subunit of the N-methyl-D-aspartate receptor (NMDAR), are linked to several forms of epileptic encephalopathy (EE). EE is a group of devastating epilepsies, which often involve intractable seizures and different brain abnormalities. The prognosis for EE patients is poor, partly due to inadequate pharmacological options that dampen symptoms, but have little ability to rectify underlying aberrant neuronal circuitry. In addition, children with EE often require around-the-clock care, placing a heavy social and financial burden on primary caregivers, making the need for early intervention with circuitry-correcting therapeutics urgent and necessary. Here, I propose experiments that explore the cellular mechanisms driving epileptiform activity in patients with loss-of-function (LoF) GRIN2A mutations, a group of genetic variants that reduce the function or cell surface expression of GluN2A-containing NMDARs. Whole exome sequencing revealed that multiple patients with EE have de novo mutations in GRIN2A, with a prevalence of roughly 1 in 80,000. Evaluation for functional activity has revealed that 56% of epilepsy-related GRIN2A mutations are LoF variants, displaying diminished receptor function and/or decreased surface expression. These results are intriguing as one might hypothesize that the loss of excitatory synaptic GluN2A subunit would decrease excitability, rather than promote epileptiform activity. To understand how diminished GluN2A function/expression can promote compensatory alterations that increase excitability, I will use Grin2a +/- and -/- mice as a model for loss-of-function GRIN2A variants. Preliminary behavioral data indicate that Grin2a -/- (2AKO) mice have a lower threshold for febrile-induced seizures compared to wildtype (WT) mice. In adult brain slices, 2AKO mice exhibit an increased frequency of epileptiform burst-firing activity in the CA1 region of the hippocampus compared to WT mice. These data indicate that the loss of GluN2A activity is sufficient to drive an epileptic phenotype, as observed in human patients with heterozygous LoF GRIN2A mutations. Taken together, these data suggest that 2AKO mice have an altered excitatory-to-inhibitory balance. Therefore, I hypothesize that epileptiform activity in 2AKO mice is: 1) dependent on a critical developmental window, 2) may be due to changes in postsynaptic receptor expression and hippocampal connectivity, and 3) could be modulated by aberrant forms of synaptic plasticity. Information on how and when the imbalance in excitation/inhibition is generated will help advance a cellular and temporal understanding on the mechanisms involved in epileptogenesis in LoF GRIN2A patients. Thus, these experiments could help guide specific anti-epileptic treatment options for LoF GRIN2A patients suffering from EE. Moreover, if aspects of epileptogenesis are shared across the spectrum of epileptic disorders, data obtained from this proposal may uncover overarching disease mechanisms without being restricted to just LoF GRIN2A patients.
Loss-of-function de novo mutations in the GRIN2A gene, which encodes the GluN2A subunit of the N- methyl-D-aspartate receptor (NMDAR), are linked to several forms of severe epileptic encephalopathy (EE). Here, I propose experiments aimed at uncovering the cellular and temporal mechanisms driving epileptic activity in patients will loss-of-function GRIN2A mutations. These experiments may elucidate a critical window for therapeutic intervention, potentially preventing pro-epileptic circuitry connections from being established.
