Activity-Dependent Alternative Splicing Increases Persistent Sodium Current and Promotes Seizure

Abstract

Activity of voltage-gated Na channels (Na v ) is modified by alternative splicing. However, whether altered splicing of human Na v s contributes to epilepsy remains to be conclusively shown. We show here that altered splicing of the Drosophila Na v (paralytic, DmNa v ) contributes to seizure-like behavior in identified seizure mutants. We focus attention on a pair of mutually exclusive alternate exons (termed K and L), which form part of the voltage sensor (S4) in domain III of the expressed channel. The presence of exon L results in a large, non-inactivating, persistent I Nap . Many forms of human epilepsy are associated with an increase in this current. In wild-type (WT) Drosophila larvae, ϳ70 -80% of DmNa v transcripts contain exon L, and the remainder contain exon K. Splicing of DmNa v to include exon L is increased to ϳ100% in both the slamdance and easily-shocked seizure mutants. This change to splicing is prevented by reducing synaptic activity levels through exposure to the antiepileptic phenytoin or the inhibitory transmitter GABA. Conversely, enhancing synaptic activity in WT, by feeding of picrotoxin is sufficient to increase I Nap and promote seizure through increased inclusion of exon L to 100%. We also show that the underlying activity-dependent mechanism requires the presence of Pasilla, an RNA-binding protein.Finally, we use computational modeling to show that increasing I Nap is sufficient to potentiate membrane excitability consistent with a seizure phenotype. Thus, increased synaptic excitation favors inclusion of exon L, which, in turn, further increases neuronal excitability. Thus, at least in Drosophila, this self-reinforcing cycle may promote the incidence of seizure.

Epilepsy research methods update: Understanding the causes of epileptic seizures and identifying new treatments using non-mammalian model organisms

“…Electrophysiological analysis in such larvae revealed enhancement of synaptic activity in motor circuitry and, importantly, increased inclusion of exon L in expressed Na v transcripts. Conversely, seizure behaviour was reduced in sda larvae following feeding with GABA, an effect that is associated with both decreased synaptic activity and reduction of inclusion of exon L. 51 Taken together, these observations indicate that the mechanism regulating the inclusion of exon L, at the expense of exon K, is activity-dependent. Moreover, increasing activity promotes inclusion of exon L, which results in larger I Nap which further promotes cell excitability.…”

“…By contrast, the inclusion of L is increased to $100% in sda. 51 Heterologous expression of K-or L-containing splice variants in Xenopus oocytes shows that these exons have a marked influence on the magnitude of the I Nap current expressed. When present, exon L results in channels with a significantly larger I Nap .…”

“…Accordingly, prior ingestion of phenytoin, which is sufficient to rescue seizure, also reversed the increased inclusion of exon L in sda back to a wild-type level. 51 Ingestion of proconvulsants (e.g. picrotoxin) by wild-type larvae is sufficient to induce a seizure phenotype.…”

“…Moreover, increasing activity promotes inclusion of exon L, which results in larger I Nap which further promotes cell excitability. 51 This positive feedback loop provides a possible mechanistic explanation of the clinical observation in which small-scale paroxysmal activity, if left untreated, can directly promote subsequent seizures. 52 Pasilla (Ps), a KH domain-containing RNA binding protein, was shown to influence the splicing balance between exons K and L. 53 The inclusion of exon K is significantly increased in a Ps loss-offunction mutant, indicating that the presence of Ps is necessary for the inclusion of exon L. 50 Thus, treatments that reduce the activity of Ps should, theoretically, be antiepileptic.…”

“…Knockdown of Ps, either by loss of one gene copy (ps +/À ) or expression of psspecific RNAi, was sufficient to diminish both the increased inclusion of exon L and seizure severity attributable to the sda mutation. 51 Homologues of Ps in mammals are the neurooncological ventral antigen 1 and 2 splice factors (Nova-1 and Nova-2, respectively). The genomic region in and around exons K and L contain several putative [T/C]CA[T/C] Nova-recognition motifs.…”