| dc.description.abstract |
The rare epilepsy disorders Epilepsy of Infancy with Migrating Focal Seizures (EIMFS) and Autosomal Dominant Sleep-related Hypermotor Epilepsy (AD)SHE are characterized by early onset of severe seizures. Both disorders are refractory to anti-epileptic drugs and associated with mutations in KCNT1, the gene encoding the sodium (Na+) activated potassium- (K+) channel Slack. To date, approximately 60 pathogenic mutations associated with EIMFS or (AD)SHE had been identified in KCNT1, most of which cause a gain-of-function (GOF), resulting in increased Slack K+ currents. While these GOF mutations result in overexcited nervous tissue, high network activity and disturbed electrical rhythms, also a loss-of-function Slack variant has been linked to epilepsies in one patient and increased neuronal vulnerability in vivo and in vitro.
As Slack crucially modulates neuronal excitability, we investigated its role during epileptic seizures using wildtype (Slack+/+) and global Slack knock-out (Slack-/-) mice in a Kainic acid (KA)-induced model of acute epilepsy. Compared to Slack+/+, 4-week-old juvenile and 12 week old adult Slack-/- mice displayed increased seizure scores and mortality after KA application, indicating a neuroprotective role of Slack during epileptic seizures in vivo. Strikingly, 24 hours after seizure onset, hippocampal GFAP, GluA and GluN mRNA levels are equal between KA-treated Slack+/+ and Slack-/-, suggesting a more prominent role for Slack during acute seizures. In addition to these findings in vivo, organotypic Slack+/+ and Slack-/- hippocampal slice cultures were exposed to KA and cell death was quantified by propidium iodide uptake. Compared to Slack+/+, sclerosis like neuronal cell loss was significantly increased in the CA regions of hippocampal slices derived from Slack-/- brains, confirming our in vivo results of KA induced seizures in mice. To confirm these results independent of hippocampal circuitries, dissociated hippocampal neurons were treated with KA. Conformingly, compared to Slack+/+, also individual Slack /- neurons responded with increased cell death to KA treatment. To elucidate whether these differences in neuronal vulnerability are caused by a differential subcellular ion homeostasis, we next assessed intracellular Ca2+ and K+ dynamics in dissociated primary hippocampal neurons derived from Slack+/+ and Slack-/- mice in response to KA treatment. Interestingly, compared with Slack+/+, Slack-/- hippocampal neurons showed increased intracellular Ca2+ dynamics in response to low but not high KA concentrations, which was not modulated by AMPA (NBQX) or NMDA receptor blockers (DL-AP5) or the “Slack activator” Loxapine. This suggests that the neuroprotective effect of Slack is mediated on the level of altered thresholds for neuronal activation patterns. Next, we employed a K+ sensitive FRET-sensor to record intracellular K+ dynamics in these primary hippocampal neurons among KA-exposure. Unexpectedly, under these conditions, KA induced K+ efflux was significantly increased in Slack-/- compared to Slack+/+ hippocampal neurons. Finally, patch-clamp recordings identified amplified TTX sensitive transient and persistent inward currents in Slack-/- neurons compared to Slack+/+. Fittingly, KA and current provoked AP firing was accelerated in current-clamped Slack-/- compared to Slack-/- neurons owing to faster accessibility of the AP threshold, increased AP rise slope and shortened after-hyperpolarization.
Based on these findings, we propose that loss of Slack leads to increased neuronal response to glutamatergic signals, ultimately causing detrimental epileptiform activity. It may additionally involve alterations of other K+ channel activities that facilitate reactivation of, for instance, voltage-gated (TTX-sensitive) Na+ channels to subsequently drive KA induced overexcitability in Slack-/- neurons. We conclude that a careful modulation of Slack activity is necessary for otherwise untreatable epilepsy syndromes. |
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