Molecular Pharmacology of Selective NaV1.6 and Dual NaV1.6/NaV1.2 Channel Inhibitors that Suppress Excitatory Neuronal Activity Ex Vivo.

Voltage-gated sodium channel (NaV) inhibitors are used to treat neurological disorders of hyperexcitability such as epilepsy. These drugs act by attenuating neuronal action potential firing to reduce excitability in the brain. However, all currently available NaV-targeting antiseizure medications nonselectively inhibit the brain channels NaV1.1, NaV1.2, and NaV1.6, which potentially limits the efficacy and therapeutic safety margins of these drugs. Here, we report on XPC-7724 and XPC-5462, which represent a new class of small molecule NaV-targeting compounds. These compounds specifically target inhibition of the NaV1.6 and NaV1.2 channels, which are abundantly expressed in excitatory pyramidal neurons. They have a > 100-fold molecular selectivity against NaV1.1 channels, which are predominantly expressed in inhibitory neurons. Sparing NaV1.1 preserves the inhibitory activity in the brain. These compounds bind to and stabilize the inactivated state of the channels thereby reducing the activity of excitatory neurons. They have higher potency, with longer residency times and slower off-rates, than the clinically used antiseizure medications carbamazepine and phenytoin. The neuronal selectivity of these compounds is demonstrated in brain slices by inhibition of firing in cortical excitatory pyramidal neurons, without impacting fast spiking inhibitory interneurons. XPC-5462 also suppresses epileptiform activity in an ex vivo brain slice seizure model, whereas XPC-7224 does not, suggesting a possible requirement of Nav1.2 inhibition in 0-Mg2+- or 4-AP-induced brain slice seizure models. The profiles of these compounds will facilitate pharmacological dissection of the physiological roles of NaV1.2 and NaV1.6 in neurons and help define the role of specific channels in disease states. This unique selectivity profile provides a new approach to potentially treat disorders of neuronal hyperexcitability by selectively downregulating excitatory circuits.

A more efficient conditional mouse model of Dravet syndrome: Implications for epigenetic selection and sex-dependent behaviors.

BACKGROUND: Dravet Syndrome (DS) is an epileptic disorder characterized by spontaneous and thermally-induced seizures, hyperactivity, cognitive deficits, autistic-like behaviors, and Sudden Unexpected Death in Epilepsy (SUDEP). DS is caused by de novo loss-of-function mutations in the SCN1A gene. Selective loss of GABAergic interneuron excitability is the primary cause of the disease. Up to 60% of Scn1a+/- mice die from SUDEP before sexual maturity. NEW METHOD: We used Cre-Lox technology to conditionally delete Scn1a in all epiblast-derived somatic cells by crossing a floxed Scn1a mouse with a mouse expressing Cre under the Meox2 promoter. RESULTS: Parental Scn1a flox (F) mice, parental Meox2 Cre+ mice, and their F/+:Meox2-Cre- offspring were phenotypically normal and did not prematurely die. In contrast, F/+:Meox2-Cre+ offspring recapitulated DS seizure and behavioral phenotypes. Unexpectedly, male F/+:Meox2-Cre+ mice demonstrated impaired social interaction, while females did not. COMPARISON WITH EXISTING METHOD: In the previous models, colony maintenance required breeding SUDEP survivors, which greatly increased colony size required to sustain experimental animal production, and raised the concern that surviving breeders have epigenetic traits that impart new phenotypes to their offspring. Our method greatly facilitates breeding, recapitulates DS phenotypes, eliminates concerns about parents that are survivors, and provides initial evidence for unexpected sex-dependent social interaction impairment. CONCLUSIONS: We introduce a more efficient mouse model of human DS that demonstrates an efficient breeding strategy free from potential inherited epigenetic changes and reveals an unexpected sex-specific impairment of social interaction in DS. This new model should have great value to investigators of DS.

Antiepileptic action of c-Jun N-terminal kinase (JNK) inhibition in an animal model of temporal lobe epilepsy.

Several phosphorylation signaling pathways have been implicated in the pathogenesis of epilepsy arising from both genetic causes and acquired insults to the brain. Identification of dysfunctional signaling pathways in epilepsy may provide novel targets for antiepileptic therapies. We previously described a deficit in phosphorylation signaling mediated by p38 mitogen-activated protein kinase (p38 MAPK) that occurs in an animal model of temporal lobe epilepsy, and that produces neuronal hyperexcitability measured in vitro. We asked whether in vivo pharmacological manipulation of p38 MAPK activity would influence seizure frequency in chronically epileptic animals. Administration of a p38 MAPK inhibitor, SB203580, markedly worsened spontaneous seizure frequency, consistent with prior in vitro results. However, anisomycin, a non-specific p38 MAPK activator, significantly increased seizure frequency. We hypothesized that this unexpected result was due to activation of a related MAPK, c-Jun N-terminal kinase (JNK). Administration of JNK inhibitor SP600125 significantly decreased seizure frequency in a dose-dependent manner without causing overt behavioral abnormalities. Biochemical analysis showed increased JNK expression and activity in untreated epileptic animals. These results show for the first time that JNK is hyperactivated in an animal model of epilepsy, and that phosphorylation signaling mediated by JNK may represent a novel antiepileptic target.

Protein kinase C bidirectionally modulates Ih and hyperpolarization-activated cyclic nucleotide-gated (HCN) channel surface expression in hippocampal pyramidal neurons.

KEY POINTS: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, particularly that of the HCN1 isoform, are enriched in the distal dendrites of hippocampal CA1 pyramidal neurons; these channels have physiological functions with respect to decreasing neuronal excitability. In the present study, we aimed to investigate phosphorylation as a mechanism controlling Ih amplitude and HCN1 surface expression in hippocampal principal neurons under normal physiological conditions. Tyrosine phosphorylation decreased Ih amplitude at maximal activation (maximal Ih ), without altering HCN1 surface expression, in two classes of hippocampal principal neurons. Inhibition of serine/threonine protein phosphatases 1 and 2A decreased maximal Ih and HCN1 surface expression in hippocampal principal neurons. Protein kinase C (PKC) activation irreversibly diminished Ih and HCN1 surface expression, whereas PKC inhibition augmented Ih and HCN1 surface expression. PKC activation increased HCN1 channel phosphorylation. These results demonstrate the novel finding of a phosphorylation mechanism, dependent on PKC activity, which bidirectionally modulates Ih amplitude and HCN1channel surface expression in hippocampal principal neurons under normal physiological conditions. ABSTRACT: Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels attenuate excitability in hippocampal pyramidal neurons. Loss of HCN channel-mediated current (Ih ), particularly that mediated by the HCN1 isoform, occurs with the development of epilepsy. Previously, we showed that, following pilocarpine-induced status epilepticus, there are two independent changes in HCN function in dendrites: decreased Ih amplitude associated with a loss of HCN1 surface expression and a hyperpolarizing shift in voltage-dependence of activation (gating). The hyperpolarizing shift in gating was attributed to decreased phosphorylation as a result of a loss of p38 mitogen-activated protein kinase activity and increased calcineurin activity; however, the mechanisms controlling Ih amplitude and HCN1 surface expression under epileptic or normal physiological conditions are poorly understood. We aimed to investigate phosphorylation as a mechanism regulating Ih amplitude and HCN1 surface expression (i.e. as is the case for HCN gating) in hippocampal principal neurons under normal physiological conditions. We discovered that inhibition of either tyrosine phosphatases or the serine/threonine protein phosphatases 1 and 2A decreased Ih at maximal activation in hippocampal CA1 pyramidal dendrites and pyramidal-like principal neuron somata from naïve rats. Furthermore, we found that inhibition of PP1/PP2A decreased HCN1 surface expression, whereas tyrosine phosphatase inhibition did not. Protein kinase C (PKC) activation reduced Ih amplitude and HCN1 surface expression, whereas PKC inhibition produced the opposite effect. Inhibition of protein phosphatases 1 and 2A and activation of PKC increased the serine phosphorylation state of the HCN1 protein. The effect of PKC activation on Ih was irreversible. These results indicate that PKC bidirectionally modulates Ih amplitude and HCN1 surface expression in hippocampal principal neurons.

Guidelines for return to duty (play) after heat illness: a military perspective.

Since Biblical times, heat injuries have been a major focus of military medical personnel. Heat illness accounts for considerable morbidity during recruit training and remains a common cause of preventable nontraumatic exertional death in the United States military. This brief report describes current regulations used by Army, Air Force, and Navy medical personnel to return active duty warfighters who are affected by a heat illness back to full duty. In addition, a description of the profile system used in evaluating the different body systems, and how it relates to military return to duty, are detailed. Current guidelines require clinical resolution, as well as a profile that that protects a soldier through repeated heat cycles, prior to returning to full duty. The Israeli Defense Force, in contrast, incorporates a heat tolerance test to return to duty those soldiers afflicted by heat stroke, which is briefly described. Future directions for U.S. military medicine are discussed.