The Voltage-gated Sodium Channel Isoform Nav1.6 Facilitates Neuronal Hyperexcitability in Genetic and Acquired Epilepsies

Voltage-gated sodium channels play a crucial role in regulating neuronal activity in the brain. One of these voltage-gated sodium channel isoforms in particular, Nav1.6, is instrumental in the initiation and propagation of action potentials that is necessary for normal brain function. Nav1.6 is highly expressed at the distal axon initial segment and the nodes of Ranvier, where the channel’s more hyperpolarized activation allows for a lower action potential threshold. In both genetic and acquired epilepsies, proexcitatory alterations to Nav1.6 can induce neuronal hyperexcitability. This hyperexcitability causes the rewiring of key neural circuits in a way that is conducive to the initiation and propagation of spontaneous seizures. Here we explore how proexcitatory changes to Nav1.6 contribute to neuronal hyperexcitability in the genetic epilepsy syndrome, early infantile epileptic encephalopathy 13 (EIEE13), and the acquired temporal lobe epilepsy (TLE).

EIEE13 is a rare neurological disorder that results predominantly from missense, de novo mutations to the SCN8A gene, which encodes Nav1.6. Patients with EIEE13 experience seizures beginning from birth to 18 months of age that are often refractory to clinically available treatments. In addition to seizures, EIEE13 patients suffer from mild to severe intellectual disability, developmental delay, and movement disorders. Here we are the first to electrophysiologically characterize novel, recurrent SCN8A mutations that account for 20% of all EIEE13 mutations known to date. As previously mentioned, patients suffering from EIEE13 are often refractory to clinically available antiepileptic drugs and literature detailing the efficacy of various treatments is sparse. To fill this knowledge gap, we test the in vitro efficacy of the sodium channel blocker phenytoin on a novel gain-of-function SCN8A mutation. We show that phenytoin shows preferential block of mutant Nav1.6 channels over wild type and that phenytoin can help decrease the proexcitatory alterations in channel function that are induced by the mutation. To grasp a better understanding of the phenotypes that result from SCN8A mutations, we also are the first to characterize novel loss-of-function SCN8A mutations from patients with intellectual disability and no seizures. This work contributes to the SCN8A field by demonstrating the spectrum of phenotypes that can result from Nav1.6 mutations and it makes a stronger case for the screening of SCN8A mutations in patients with idiopathic intellectual disorders.

To grasp a deeper understanding of EIEE13 that goes beyond in vitro cultured cell experiments, a mouse model of EIEE13 was generated by knocking in the p.Asn1768Asp (N1768D) mutation. Mice carrying this mutation recapitulate many of the phenotypes seen in human patients including motor disorder, seizures, and SUDEP. To better understand how the N1768D mutation contributes to neuronal hyperexcitability, we electrophysiologically characterized layer II excitatory neurons of the medial entorhinal cortex (mEC) of heterozygote (D/+) and homozygote (D/D) mice. Layer II mEC neurons from D/+ and D/D are hyperexcitable in response to somatic current injection steps and synaptic stimulation. Additionally, D/+ and D/D mice show a dose-dependent increase in both persistent (INaP) and resurgent (INaR) sodium currents, two currents that strongly contribute to bursting and high frequency action potential firing. Our work using the N1768D mouse model further expands our knowledge of this rare and devastating disorder and it paves the way for future, more advanced in vitro and in vivo experiments.
Temporal lobe epilepsy (TLE) is one of the most common forms of adult epilepsy and it is marked by seizures that originate from the temporal lobe. While seizure-induced changes in the dentate gyrus (DG) and Cornu Ammonis (CA) regions have been well studied, relatively little attention has been given to alterations that occur in the subiculum, a region that acts as an output of the temporal lobe. This gap is literature is surprising as the subiculum consists of a large population of endogenously bursting neurons and is largely spared in TLE. Here we explore the neuronal excitability in subiculum excitatory neurons, and how it is altered in TLE. Specifically, we see that TLE subiculum bursting neurons are hyperexcitable in response to somatic current injection steps and synaptic stimulation. We hypothesized that alterations in sodium channel physiology contribute to this neuronal hyperexcitability seen in the subiculum in TLE. Outside-out patch clamp recordings revealed proexcitatory alterations in sodium channel physiology from TLE neurons compared to wild type. Additionally, we saw significant increases in both persistent (INaP) and resurgent (INaR) sodium channel currents in TLE subiculum neurons compared to controls. We believed that proexcitatory changes to Nav1.6 in particular were the predominant driver of subiculum neuronal hyperexcitability in TLE. To test this hypothesis we applied the TTX metabolite 4,9-anhydro-tetrodotoxin (4,9-ah-TTX), which has higher affinity for Nav1.6 over other sodium channel isoforms, to TLE subiculum neurons. We saw that 4,9-ah-TTX significantly reduced TLE subiculum neuron hyperexcitability and caused significant reductions in both INaP and INaR currents. These findings shed more light on the intrinsic changes that occur in TLE and expand our existing knowledge of the cellular and synaptic reorganization that occurs in the disorder. Additionally, this data makes a stronger case to continue the push to develop the elusive Nav1.6 selective blocker, a drug that could be the next promising treatment option for patients suffering from TLE.