by Jonathan E. Kurz, MD, PhD, and Dane M. Chetkovich, MD, PhD. References. 1. ... Veeramah KR, O'Brien JE, Meisler MH, Chen X, Dib-Hajj SD, Waxman.
Current Literature In Basic Science
Understanding Network Connections Connects Genotype to Epilepsy Phenotype
Regulation of Thalamic and Cortical Network Synchrony by Scn8a Makinson CD, Tanaka BS, Sorokin JM, Wong JC, Christian CA, Goldin AL, Escayg A, Huguenard JR. Neuron 2017;93:1165– 1179.
Voltage-gated sodium channel (VGSC) mutations cause severe epilepsies marked by intermittent, pathological hypersynchronous brain states. Here we present two mechanisms that help to explain how mutations in one VGSC gene, Scn8a, contribute to two distinct seizure phenotypes: (1) hypoexcitation of cortical circuits leading to convulsive seizure resistance, and (2) hyperexcitation of thalamocortical circuits leading to non-convulsive absence epilepsy. We found that loss of Scn8a leads to altered RT cell intrinsic excitability and a failure in recurrent RT synaptic inhibition. We propose that these deficits cooperate to enhance thalamocortical network synchrony and generate pathological oscillations. To our knowledge, this finding is the first clear demonstration of a pathological state tied to disruption of the RT-RT synapse. Our observation that loss of a single gene in the thalamus of an adult wild-type animal is sufficient to cause spike-wave discharges is striking and represents an example of absence epilepsy of thalamic origin.
Commentary Voltage-gated sodium channels (VGSCs) play a critical role in the propagation of electrical signaling in neurons, providing for the rapid and regenerative inward current seen during the rising phase of the action potential. Four of the nine known VGSC alpha subunit genes are primarily expressed in the central nervous system (SCN1A, SCN2A, SCN3A, SCN8A). Mutations in each of these genes have been implicated in genetic epilepsies of varying phenotypes, ranging in severity from Dravet syndrome to generalized epilepsy with febrile seizures plus (GEFS+) to simple febrile seizures. Initial studies of homozygous knockout of Scn8a in the mouse demonstrated a phenotype of ataxia and cerebellar tremor, while initial human cases of SCN8A similarly described ataxia, cerebellar atrophy, and intellectual disability (1). More recently, investigations into heterozygous Scn8a knockout in mice have revealed an intriguing dual role for the channel in epilepsy. The biophysical properties of NaV1.6, the sodium channel alpha subunit encoded by SCN8A, allow these channels to play a unique role in regulating neuronal excitability. NaV1.6 is involved in producing the persistent current, a steady-state sodium current that is involved in the initiation of action potentials at near-threshold voltages, and the resurgent current (2, 3), a small, transient current elicited by depolarization after an action potential that facilitates rapid, repetitive neuronal firing. Neurons expressing NaV1.6 channels are more excitable Epilepsy Currents, Vol. 17, No. 4 (July/August) 2017 pp. 239–240 © American Epilepsy Society
than those containing only NaV1.1 and 1.2, and loss of NaV1.6 is thus associated with a higher threshold for the initiation of action potentials (3). These properties may help explain why loss of NaV1.6 function is associated with increased seizure resistance in rodent models; heterzygosity for null or missense mutations of Scn8a produces a resistance to chemically induced seizures (4) and amygdala kindling (5). Additionally, SCN8A mutations can modify the severity of epileptic phenotypes associated with other sodium channel mutations. For example, the reduced seizure threshold and early mortality seen in Scn1a-knockout mice is rescued by coexpression of a Scn8a null allele (6). Human gain of function mutations in SCN8A have been identified in children with epileptic encephalopathy (7), while hippocampal kindling in mice is associated with increased hippocampal expression of NaV1.6 (5), further suggesting that NaV1.6 channel expression can exert a proexcitatory effect. These findings could make NaV1.6 inhibition a promising target for the development of future anticonvulsants, with potential to even have benefit in other sodium channelopathy-associated epilepsies, such as Dravet syndrome. However, further investigations have shown another side to the story. While Scn8amed/+ (loss of function) and Scn8amed-jo/+ (a missense mutation with altered voltage gating) heterozygotes reduced susceptibility to induced seizures, both lines of mice also exhibited episodes of well-defined, ethosuximidesensitive spontaneous spike-wave discharges that were associated with immobility – a rodent analog of absence epilepsy (8). Subsequently, a patient with treatment-resistant absence epilepsy has been identified with a complex combination of a heterozygous SCN8A missense mutation and mosaic dele-
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Connecting Genotype to Epilepsy Phenotype
tion of a portion of the SCN8A gene (9). The fact that loss of function mutations in a single gene can produce dichotomous outcomes on two distinct types of epilepsy strongly suggests a cell-type or circuit-specific role of NaV1.6-mediated sodium currents; however, the details of how these channels differentially affect network excitability were previously unclear. These are the questions that Makinson et al. convincingly explore in this article: understanding the role of NaV1.6 expression in specific populations of excitatory and inhibitory neurons in seizure susceptibility and investigating the mechanism of NaV1.6-associated absence epilepsy. Using heterozygous cell-type specific knockout of Scn8a, the authors first examined the effect of decreasing NaV1.6 expression broadly in excitatory neurons; in inhibitory neurons, except those of the thalamic reticular nucleus (RT); or broadly across most inhibitory neurons. Recapitulating results from global heterozygous Scn8a null allele expression, heterozygous knockout in excitatory neurons reduced susceptibility to flurothyl-induced seizures, increased latency to epileptiform bursts in an in vitro preparation, and mitigated the severity of an Scn1a knockout (GEFS+ like) phenotype. Conversely, broad knockout of Scn8a in inhibitory cells did not reduce seizure susceptibility but did produce absence-like spike-wave discharges (SWD) on electrocorticography. SWD were not seen in animals with excitatory-cell specific knockout of Scn8a, nor were they seen if knockout was restricted to inhibitory cells outside of the RT. Double knockout animals lacking Scn8a from both excitatory and inhibitory cell lines exhibited a lower frequency of SWD. Overall, the authors suggest that loss of Scn8a from excitatory neurons lowers cortical excitability, reducing seizure susceptibility and opposing the absence seizure generation that is driven by loss of Scn8a from inhibitory cells. As a manifestation of the hypersynchronous thalamocortical oscillations that are thought to underlie absence epilepsy, Makinson et al. demonstrated that loss of Scn8a in inhibitory neurons of the thalamus led to an increase in both spontaneous and evoked oscillatory activity in an in vivo thalamic slice preparation and drove RT neurons toward a burst, rather than tonic, firing mode. Using optogenetic stimulation of RT cells, with concurrent intracellular measurement of evoked inhibitory postsynaptic potentials in thalamocortical (TC) or neighboring RT neurons, the authors were able to demonstrate that loss of Scn8a impairs intra-RT inhibition but not RT-TC inhibition. Intra-RT inhibitory activity is thought to constrain TC oscillatory activity, with loss of this inhibition proposed as a cause of hypersynchrony, thalamic dysfunction, and subsequent epilepsy (10). Further confirming the importance of Scn8a for the maintenance of normal RT function, short hairpin RNA(shRNA)-mediated knockdown of Scn8a, delivered by a viral vector into the RT, was sufficient to induce frequent absence seizures. On the other hand, shRNA delivery to TC neurons alone did not induce absence epilepsy, nor did expression of a scrambled RNA unable to knockdown Scn8a. Overall the findings are a novel demonstration of Scn8a’s regulation of thalamic synchrony via RT firing state and intraRT inhibition and fit well within a framework where absence seizures result from a perturbation of normal thalamic oscil-
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latory activity. The authors present a plausible mechanism by which knockout of NaV1.6 simultaneously results in cortical hypoexcitability and thalamic hypersynchrony, which may inform our understanding of the phenotype associated with newly identified SCN8A mutations. Unfortunately, this dual role may complicate any effort to take advantage of systemic pharmacologic manipulation of NaV1.6 as an anticonvulsant. Beyond expanding our understanding of the thalamic mechanisms underlying the generation of absence epilepsy, the role of NaV1.6 channels in the regulation of thalamic oscillatory activity may have broader significance related to the regulation of sleep, consciousness and attention, which should drive future studies of interest into the function of this channel. by Jonathan E. Kurz, MD, PhD, and Dane M. Chetkovich, MD, PhD References 1. Trudeau MM, Dalton JC, Day JW, Ranum LPW, Meisler MH. Heterozygosity for a protein truncation mutation of sodium channel SCN8A in a patient with cerebellar atrophy, ataxia, and mental retardation. J Med Genet 2006;43:527–530. doi:10.1136/jmg.2005.035667. 2. Raman IM, Sprunger LK, Meisler MH, Bean BP. Altered subthreshold sodium currents and disrupted firing patterns in Purkinje neurons of Scn8a mutant mice. Neuron 1997;19:881–891. doi:10.1016/S08966273(00)80969-1. 3. O’Brien JE, Meisler MH. Sodium channel SCN8A (Nav1.6): properties and de novo mutations in epileptic encephalopathy and intellectual disability. Front Genet 2013;4:1–9. doi:10.3389/fgene.2013.00213. 4. Makinson CD, Tanaka BS, Lamar T, Goldin AL, Escayg A. Role of the hippocampus in Nav1.6 (Scn8a) mediated seizure resistance. Neurobiol Dis 2014;68:16–25. doi:10.1016/j.nbd.2014.03.014. 5. Blumenfeld H, Lampert A, Klein JP, Mission J, Chen MC, Rivera M, Dib-Hajj S, Brennan AR, Hains BC, Waxman SG. Role of hippocampal sodium channel Nav1.6 in kindling epileptogenesis. Epilepsia 2009;50:44–55. doi:10.1111/j.1528-1167.2008.01710.x. 6. Hawkins NA, Martin MS, Frankel WN, Kearney JA, Escayg A. Neuronal voltage-gated ion channels are genetic modifiers of generalized epilepsy with febrile seizures plus. Neurobiol Dis 2011;41:655–660. doi:10.1016/j.nbd.2010.11.016. 7. Veeramah KR, O’Brien JE, Meisler MH, Chen X, Dib-Hajj SD, Waxman SG, Talwar D, Girirajan S, Eichler EE, Restifo LL, Erickson RP, Hammer MF. De novo pathogenic SCN8A mutation identified by wholegenome sequencing of a family quartet affected by infantile epileptic encephalopathy and SUDEP. Am J Hum Genet 2012;90:502–510. doi:10.1016/j.ajhg.2012.01.006. 8. Papale LA, Beyer B, Jones JM, Sharkey LM, Tufik S, Epstein M, Letts VA, Meisler MH, Frankel WN, Escayg A. Heterozygous mutations of the voltage-gated sodium channel SCN8A are associated with spike-wave discharges and absence epilepsy in mice. Hum Mol Genet 2009;18:1633–1641. doi:10.1093/hmg/ddp081. 9. Berghuis B, de Kovel CGF, van Iterson L, Lamberts RJ, Sander JW, Lindhout D, Koeleman BP. Complex SCN8A DNA-abnormalities in an individual with therapy resistant absence epilepsy. Epilepsy Res 2015;115:141–144. doi:10.1016/j.eplepsyres.2015.06.007. 10. Beenhakker MP, Huguenard JR. Neurons that fire together also conspire together: is normal sleep circuitry hijacked to generate epilepsy? Neuron 2009;62:612–632. doi:10.1016/j.neuron.2009.05.015.
