BackgroundDravet syndrome is a devastating infantile-onset epilepsy syndrome with cognitive deficits and autistic traits caused by genetic alterations in SCN1A gene encoding the α-subunit of the voltage-gated sodium channel Nav1.1. Disease modeling using patient-derived induced pluripotent stem cells (iPSCs) can be a powerful tool to reproduce this syndrome’s human pathology. However, no such effort has been reported to date. We here report a cellular model for DS that utilizes patient-derived iPSCs.ResultsWe generated iPSCs from a Dravet syndrome patient with a c.4933C>T substitution in SCN1A, which is predicted to result in truncation in the fourth homologous domain of the protein (p.R1645*). Neurons derived from these iPSCs were primarily GABAergic (>50%), although glutamatergic neurons were observed as a minor population (<1%). Current-clamp analyses revealed significant impairment in action potential generation when strong depolarizing currents were injected.ConclusionsOur results indicate a functional decline in Dravet neurons, especially in the GABAergic subtype, which supports previous findings in murine disease models, where loss-of-function in GABAergic inhibition appears to be a main driver in epileptogenesis. Our data indicate that patient-derived iPSCs may serve as a new and powerful research platform for genetic disorders, including the epilepsies.

Exposure to maternal stress (MS) and mutations in GAD1, which encodes the γ-aminobutyric acid (GABA) synthesizing enzyme glutamate decarboxylase (GAD) 67, are both risk factors for psychiatric disorders. However, the relationship between these risk factors remains unclear. Interestingly, the critical period of MS for psychiatric disorders in offspring corresponds to the period of GABAergic neuron neurogenesis and migration in the fetal brain, that is, in the late stage of gestation. Indeed, decrement of parvalbumin (PV)-positive GABAergic interneurons in the medial prefrontal cortex (mPFC) and hippocampus (HIP) has often been observed in schizophrenia patients. In the present study, we used GAD67-green fluorescent protein (GFP) knock-in mice (that is, mice in which the Gad1 gene is heterozygously deleted; GAD67+/GFP) that underwent prenatal stress from embryonic day 15.0 to 17.5 and monitored PV-positive GABAergic neurons to address the interaction between Gad1 disruption and stress. Administration of 5-bromo-2-deoxyuridine revealed that neurogenesis of GFP-positive GABAergic neurons, but not cortical plate cells, was significantly diminished in fetal brains during MS. Differential expression of glucocorticoid receptors by different progenitor cell types may underlie this differential outcome. Postnatally, the density of PV-positive, but not PV-negative, GABAergic neurons was significantly decreased in the mPFC, HIP and somatosensory cortex but not in the motor cortex of GAD67+/GFP mice. By contrast, these findings were not observed in wild-type (GAD67+/+) offspring. These results suggest that prenatal stress, in addition to heterozygous deletion of Gad1, could specifically disturb the proliferation of neurons destined to be PV-positive GABAergic interneurons.

Cortical GABAergic interneurons originate from ganglionic eminences and tangentially migrate into the cortical plate at early developmental stages. To elucidate the characteristics of this migration of GABAergic interneurons in living animals, we established an experimental design specialized for in vivo time-lapse imaging of the neocortex of neonate mice with two-photon laser-scanning microscopy. In vesicular GABA/glycine transporter (VGAT)-Venus transgenic mice from birth (P0) through P3, we observed multidirectional tangential migration of genetically-defined GABAergic interneurons in the neocortical marginal zone. The properties of this migration, such as the motility rate (distance/hr), the direction moved, and the proportion of migrating neurons to stationary neurons, did not change through P0 to P3, although the density of GABAergic neurons at the marginal zone decreased with age. Thus, the characteristics of the tangential motility of individual GABAergic neurons remained constant in development. Pharmacological block of GABAA receptors and of the Na+-K+-Cl− cotransporters, and chelating intracellular Ca2+, all significantly reduced the motility rate in vivo. The motility rate and GABA content within the cortex of neonatal VGAT-Venus transgenic mice were significantly greater than those of GAD67-GFP knock-in mice, suggesting that extracellular GABA concentration could facilitate the multidirectional tangential migration. Indeed, diazepam applied to GAD67-GFP mice increased the motility rate substantially. In an in vitro neocortical slice preparation, we confirmed that GABA induced a NKCC sensitive depolarization of GABAergic interneurons in VGAT-Venus mice at P0-P3. Thus, activation of GABAAR by ambient GABA depolarizes GABAergic interneurons, leading to an acceleration of their multidirectional motility in vivo.

The hetero-tetrameric voltage-gated potassium channel Kv7.2/Kv7.3, which is encoded by KCNQ2 and KCNQ3, plays an important role in limiting network excitability in the neonatal brain. Kv7.2/Kv7.3 dysfunction resulting from KCNQ2 mutations predominantly causes self-limited or benign epilepsy in neonates, but also causes early onset epileptic encephalopathy. Retigabine (RTG), a Kv7.2/ Kv7.3-channel opener, seems to be a rational antiepileptic drug for epilepsies caused by KCNQ2 mutations. We therefore evaluated the effects of RTG on seizures in two strains of knock-in mice harboring different Kcnq2 mutations, in comparison to the effects of phenobarbital (PB), which is the first-line antiepileptic drug for seizures in neonates. The subjects were heterozygous knock-in mice (Kcnq2Y284C/+ and Kcnq2A306T/+) bearing the Y284C or A306T Kcnq2 mutation, respectively, and their wild-type (WT) littermates, at 63–100 days of age. Seizures induced by intraperitoneal injection of kainic acid (KA, 12mg/kg) were recorded using a video-electroencephalography (EEG) monitoring system. Effects of RTG on KA-induced seizures of both strains of knock-in mice were assessed using seizure scores from a modified Racine’s scale and compared with those of PB. The number and total duration of spike bursts on EEG and behaviors monitored by video recording were also used to evaluate the effects of RTG and PB. Both Kcnq2Y284C/+ and Kcnq2A306T/+ mice showed significantly more KA-induced seizures than WT mice. RTG significantly attenuated KA-induced seizure activities in both Kcnq2Y284C/+ and Kcnq2A306T/+ mice, and more markedly than PB. This is the first reported evidence of RTG ameliorating KA-induced seizures in knock-in mice bearing mutations of Kcnq2, with more marked effects than those observed with PB. RTG or other Kv7.2-channel openers may be considered as first-line antiepileptic treatments for epilepsies resulting from KCNQ2 mutations.

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Objective NF‐κB–dependent signaling is an important modulator in osteoarthritis (OA), and G protein–coupled receptor kinase 5 (GRK5) regulates the NF‐κB pathway. This study was undertaken to investigate the functional involvement of GRK5 in OA pathogenesis. Methods GRK5 expression in normal and OA human knee joints was analyzed immunohistochemically. Gain‐ or loss‐of‐function experiments were performed using human and mouse chondrocytes. OA was induced in GRK5‐knockout mice by destabilization of the medial meniscus, and histologic examination was performed. OA was also induced in wild‐type mice, which were then treated with an intraarticular injection of amlexanox, a selective GRK5 inhibitor, every 5 days for 8 weeks. Results GRK5 protein expression was increased in human OA cartilage. In vitro, expression levels of OA‐related factors and NF‐κB transcriptional activation were down‐regulated by suppression of the GRK5 gene in human OA chondrocytes (3.49‐fold decrease in IL6 [P < 0.01], 2.43‐fold decrease in MMP13 [P < 0.01], and 2.66‐fold decrease in ADAMTS4 [P < 0.01]). Conversely, GRK5 overexpression significantly increased the expression of OA‐related catabolic mediators and NF‐κB transcriptional activation. On Western blot analysis, GRK5 deletion reduced IκBα phosphorylation (up to 4.4‐fold decrease [P < 0.05]) and decreased p65 nuclear translocation (up to 6.4‐fold decrease [P < 0.01]) in mouse chondrocytes. In vivo, both GRK5 deletion and intraarticular amlexanox protected mouse cartilage against OA. Conclusion Our results suggest that GRK5 regulates cartilage degradation through a catabolic response mediated by NF‐κB signaling, and is a potential target for OA treatment. Furthermore, amlexanox may be a major compound in relevant drugs.

Water adsorption onto materials surface is known to change macroscopic surface properties such as wettability and friction coefficient. While the role of the adsorbed water has been discussed for a...