Dravet syndrome (also called severe myoclonic epilepsy of infancy) is one of the most severe forms of childhood epilepsy. Most patients have heterozygous mutations in SCN1A, encoding voltage-gated sodium channel Na v 1.1 ␣ subunits. Sodium channels are modulated by ␤1 subunits, encoded by SCN1B, a gene also linked to epilepsy. Here we report the first patient with Dravet syndrome associated with a recessive mutation in SCN1B (p.R125C). Biochemical characterization of p.R125C in a heterologous system demonstrated little to no cell surface expression despite normal total cellular expression. This occurred regardless of coexpression of Na v 1.1 ␣ subunits. Because the patient was homozygous for the mutation, these data suggest a functional SCN1B null phenotype. To understand the consequences of the lack of ␤1 cell surface expression in vivo, hippocampal slice recordings were performed in Scn1b Ϫ/Ϫ versus Scn1b ϩ/ϩ mice. Scn1b Ϫ/Ϫ CA3 neurons fired evoked action potentials with a significantly higher peak voltage and significantly greater amplitude compared with wild type. However, in contrast to the Scn1a ϩ/Ϫ model of Dravet syndrome, we found no measurable differences in sodium current density in acutely dissociated CA3 hippocampal neurons. Whereas Scn1b Ϫ/Ϫ mice seize spontaneously, the seizure susceptibility of Scn1b ϩ/Ϫ mice was similar to wild type, suggesting that, like the parents of this patient, one functional SCN1B allele is sufficient for normal control of electrical excitability. We conclude that SCN1B p.R125C is an autosomal recessive cause of Dravet syndrome through functional gene inactivation.

The ␤ subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming ␣ subunits, as well as their trafficking and localization. In heterologous expression systems, ␤1, ␤2, and ␤3 subunits influence inactivation and persistent current in different ways. To test how the ␤4 protein regulates Na channel gating, we transfected ␤4 into HEK (human embryonic kidney) cells stably expressing Na V 1.1. Unlike a free peptide with a sequence from the ␤4 cytoplasmic domain, the full-length ␤4 protein did not block open channels. Instead, ␤4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of noninactivating current. Consequently, persistent current tripled in amplitude. Expression of ␤1 or chimeric subunits including the ␤1 extracellular domain, however, favored inactivation. Coexpressing Na V 1.1 and ␤4 with ␤1 produced tiny persistent currents, indicating that ␤1 overcomes the effects of ␤4 in heterotrimeric channels. In contrast, ␤1 C121W , which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by ␤4 and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with ␤4, persistent current was slightly but significantly increased. Moreover, in ␤4-expressing neurons from Scn1b and Scn1b/Scn2b null mice, entry into inactivated states was slowed. These data suggest that ␤1 and ␤4 have antagonistic roles, the former favoring inactivation, and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted ␤1 subunits.

Voltage-gated Naϩ channel ␤1 subunits are multifunctional, participating in channel modulation and cell adhesion in vitro. We previously demonstrated that ␤1 promotes neurite outgrowth of cultured cerebellar granule neurons (CGNs) via homophilic adhesion. Both lipid raft-associated kinases and nonraft fibroblast growth factor (FGF) receptors are implicated in cell adhesion molecule-mediated neurite extension. In the present study, we reveal that ␤1-mediated neurite outgrowth is abrogated in Fyn and contactin (Cntn) null CGNs. ␤1 protein levels are unchanged in Fyn null brains, whereas levels are significantly reduced in Cntn null brain lysates. FGF or EGF (epidermal growth factor) receptor kinase inhibitors have no effect on ␤1-mediated neurite extension. These results suggest that ␤1-mediated neurite outgrowth occurs through a lipid raft signaling mechanism that requires the presence of both fyn kinase and contactin. In vivo, Scn1b null mice show defective CGN axon extension and fasciculation indicating that ␤1 plays a role in cerebellar microorganization. In addition, we find that axonal pathfinding and fasciculation are abnormal in corticospinal tracts of Scn1b null mice consistent with the suggestion that ␤1 may have widespread effects on postnatal neuronal development. These data are the first to demonstrate a cell-adhesive role for ␤1 in vivo. We conclude that voltage-gated Na ϩ channel ␤1 subunits signal via multiple pathways on multiple timescales and play important roles in the postnatal development of the CNS.

SUMMARY Vertebrate circadian rhythms are organized by the hypothalamic suprachiasmatic nucleus (SCN). Despite its physiological importance, SCN development is poorly understood. Here, we show that Lim homeodomain transcription factor 1 (Lhx1) is essential for terminal differentiation and function of the SCN. Deletion of Lhx1 in the developing SCN results in loss of SCN-enriched neuropeptides involved in synchronization and coupling to downstream oscillators, among other aspects of circadian function. Intact, albeit damped, clock gene expression rhythms persist in Lhx1-deficient SCN; however, circadian activity rhythms are highly disorganized and susceptible to surprising changes in period, phase, and consolidation following neuropeptide infusion. Our results identify a factor required for SCN terminal differentiation. In addition, our in vivo study of combinatorial SCN neuropeptide disruption uncovered synergies among SCN-enriched neuropeptides in regulating normal circadian function. These animals provide a platform for studying the central oscillator's role in physiology and cognition.

The safety and efficacy of chemotherapeutics can vary as a function of the time of their delivery during the day. This study aimed to improve the treatment of glioblastoma (GBM), the most common brain cancer, by testing whether the efficacy of the DNA alkylator temozolomide (TMZ) varies with the time of its administration. We found cell-intrinsic, daily rhythms in both human and mouse GBM cells. Circadian time of treatment affected TMZ sensitivity of murine GBM tumor cells in vitro. The maximum TMZ-induced DNA damage response, activation of apoptosis, and growth inhibition occurred near the daily peak in expression of the core clock gene Bmal1. Deletion of Bmal1 (Arntl) abolished circadian rhythms in gene expression and TMZ-induced activation of apoptosis and growth inhibition. These data indicate that tumor cell-intrinsic circadian rhythms are common to GBM tumors and can regulate TMZ cytotoxicity. Optimization of GBM treatment by timing TMZ administration to daily rhythms should be evaluated in prospective clinical trials.