Loss of the potassium channel beta-subunit gene, KCNAB2, is associated with epilepsy in patients with 1p36 deletion syndrome

PURPOSE: Clinical features associated with chromosome 1p36 deletion include characteristic craniofacial abnormalities, mental retardation, and epilepsy. The presence and severity of specific phenotypic features are likely to be correlated with loss of a distinct complement of genes in each patient. We hypothesize that hemizygous deletion of one, or a few, critical gene(s) controlling neuronal excitability is associated with the epilepsy phenotype. Because ion channels are important determinants of seizure susceptibility and the voltage-gated K(+) channel beta-subunit gene, KCNAB2, has been localized to 1p36, we propose that deletion of this gene may be associated with the epilepsy phenotype. METHODS: Twenty-four patients were evaluated by fluorescence in situ hybridization with a probe containing KCNAB2. Clinical details were obtained by neurologic examination and EEG. RESULTS: Nine patients are deleted for the KCNAB2 locus, and eight (89%) of these have epilepsy or epileptiform activity on EEG. The majority of patients have a severe seizure phenotype, including infantile spasms. In contrast, of those not deleted for KCNAB2, only 27% have chronic seizures, and none had infantile spasms. CONCLUSIONS: Lack of the beta subunit would be predicted to reduce K(+) channel-mediated membrane repolarization and increase neuronal excitability, suggesting a possible relation between loss of this gene and the development of seizures. Because some patients with seizures were not deleted for KCNAB2, there may be additional genes within 1p36 that contribute to epilepsy in this syndrome. Hemizygosity of this gene in a majority of monosomy 1p36 syndrome patients with epilepsy suggests that haploinsufficiency for KCNAB2 is a significant risk factor for epilepsy.     

下调GABA受体基因对癫痫模型大鼠海马神经细胞凋亡的影响及作用机制

目的 探讨下调GABA受体基因对癫痫模型大鼠海马神经细胞凋亡的影响及作用机制。方法 选择30只SD健康雄性大鼠,建立癫痫模型,建模成功后分为模型组、上调组、下调组各10只; 进行细胞转染建立稳定转染的GABA上调组、GABA下调组; 检测3组大鼠海马神经细胞凋亡及PI3K、AKt、mTOR、Bax、Caspase-3蛋白表达水平。结果 上调组大鼠各时间点海马神经细胞凋亡率高于模型组(P<0.05); 下调组大鼠各时间点海马神经细胞凋亡率低于模型组(P<0.05); 下调组大鼠各时间点海马神经细胞凋亡率低于上调组(P<0.05)。上调组大鼠各时间点海马神经细胞增殖率、Bcl-2、p-PI3K、p-AKt、p-mTOR蛋白表达水平均低于模型组,而Bax、Caspase-3蛋白表达水平高于模型组(P<0.05); 下调组大鼠各时间点海马神经细胞增殖率、Bcl-2、p-PI3K、p-AKt、p-mTOR蛋白表达水平均高于模型组,而Bax、Caspase-3蛋白表达水平低于模型组(P<0.05); 下调组大鼠各时间点海马神经细胞增值率、Bcl-2、p-PI3K、p-AKt、p-mTOR蛋白表达水平均高于上调组,而Bax、Caspase-3蛋白表达水平低于上调组(P<0.05)。结论 下调GABA受体基因可能是通过调节PI3K/AKt/mTOR来作用于下游凋亡靶基因,最终抑制癫痫模型大鼠海马神经细胞凋亡     

Ketogenic diet treatment abolishes seizure periodicity and improves diurnal rhythmicity in epileptic Kcna1-null mice

Introduction:   Seizures are known to perturb circadian rhythms in humans as well as in animal models of epilepsy. However, it is unknown whether treatment of the underlying epilepsy restores normal biologic rhythms. We asked whether: (1) seizure activity is characterized by diurnal rhythmicity, (2) chronically epileptic mice exhibit impaired rest–activity rhythms, and (3) treatment with the anticonvulsant ketogenic diet (KD) improves such perturbations.
Methods:   Chronically epileptic Kcna1 -null mice were fed either a standard diet (SD) or KD for 4 weeks and subjected to continuous video-EEG (electroencephalography) and actigraphy monitoring for 3–5 days to assess seizure activity and rest–activity cycles.
Results:   Seizure activity in Kcna1 -null mice demonstrated diurnal rhythmicity, peaking at zeitgeber (ZT)2.30 ± 1.52. Rest–activity rhythms of epileptic mice were significantly disrupted. Whereas locomotor activity for wild-type mice peaked at ZT15.45 ± 0.28 (ZT14:26–ZT16:51), peak activity of epileptic mice was more unpredictable, occurring over a 12.4 h range (ZT06:33–ZT18:57). In six of nine epileptic mice, peak activity was delayed to ZT17.42 ± 0.38, whereas peak activity was advanced to ZT10.00 ± 1.26 in the remaining mice. Treatment with the KD abolished seizure periodicity and restored the rest–activity rhythm to values resembling those of wild-type mice (i.e., activity peaking at ZT16.73 ± 0.67).
Conclusions:   Kcna1 -null mice experience seizures with 24-h periodicity and impaired circadian behavior. KD reduces the number and periodicity of seizures and restores normal behavioral rhythms, suggesting that this nonpharmacologic therapy may benefit biologic rhythm disturbances in epileptic patients.     

Postictal single-cell firing patterns in the hippocampus

PURPOSE: Patients with epilepsy have varying degrees of postictal impairment including confusion and amnesia. This impairment adds substantially to the disease burden of epilepsy. However, the mechanism responsible for postictal cognitive impairment is unclear. The purpose of this study was to study single-cell firing patterns in hippocampal cells after spontaneous seizures in rats previously subjected to status epilepticus. METHODS: In this study, we monitored place cells and interneurons in the CA1 region of the hippocampus before and after spontaneous seizures in six epileptic rats with a history of status epilepticus. Place cells fire action potentials when the animal is in a specific location in space, the so-called place field. Place cell function correlates well with performance in tasks of visual-spatial memory and appears to be an excellent surrogate measure of spatial memory. RESULTS: Twelve spontaneous seizures were recorded. After the seizures, a marked decrease in firing rate of action potentials from place cells was noted, whereas interneuron firing was unchanged. In addition, when place cell firing fields persisted or returned, they had aberrant firing fields with reduced coherence and information content. In addition to postictal suppression of firing patterns, seizures led to the emergence of firing fields in previously silent cells, demonstrating a postictal remapping of the hippocampus. CONCLUSIONS: These findings demonstrate that postictal alterations in behavior are not due solely to reduced neuronal firing. Rather, the postictal period is characterized by robust and dynamic changes in cell-firing patterns resulting in remapping of the hippocampal map.     

Effect of interictal spikes on single-cell firing patterns in the hippocampus

PURPOSE: The interictal EEG spike(s) is the hallmark of the epileptic EEG. While focal interictal spike (IS) have been associated with transitory cognitive impairment, with the type of deficit dependent on where in the cortex the IS arises, the mechanism by which IS result in transitory dysfunction is not known. The purpose of this study was to determine the effect of IS on single-cell firing patterns in freely moving rats with a prior history of seizures. METHODS: We studied IS in two seizure models; pilocarpine-induced status epilepticus and recurrent flurothyl models. The effect of spontaneous hippocampal spikes on action potentials (APs) of CA1 cells in rats walking in a familiar environment was investigated using 32 extracellular electrodes. We also compared the effect of spikes on two types of hippcampal cells; place cells that discharge rapidly only when the rat's head is in a specific part of the environment, the so-called firing field, and interneurons, which are a main source of inhibition in the hippocampus. RESULTS: IS were associated with a decreased likelihood of AP compared with IS-free portions of the record. Compared to pre-IS baseline, IS were followed by significant decreases in CA1 APs for periods up to 2 s following the IS in both models. When occurring in flurries, IS were associated with a pronounced decrease in APs. The response to IS was cell-dependent; IS resulted in decreases in AP firing after the IS in interneurons but not place cells. CONCLUSIONS: This study demonstrates that IS have substantial effects on cellular firing in the hippocampus and that these effects last far longer than the spike and slow wave. Furthermore, the effect of IS on cellular firing was cell specific, affecting interneurons more than place cells. These findings suggest that IS may contribute to seizure-induced cognitive impairment by altering AP firing in a cell-specific manner.     

The clinicopathologic spectrum of focal cortical dysplasias: a consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission

Purpose: Focal cortical dysplasias (FCD) are localized regions of malformed cerebral cortex and are very frequently associated with epilepsy in both children and adults. A broad spectrum of histopathology has been included in the diagnosis of FCD. An ILAE task force proposes an international consensus classification system to better characterize specific clinicopathological FCD entities. Methods: Thirty‐two Task Force members have reevaluated available data on electroclinical presentation, imaging, neuropathological examination of surgical specimens as well as postsurgical outcome. Key Findings: The ILAE Task Force proposes a three‐tiered classification system. FCD Type I refers to isolated lesions, which present either as radial (FCD Type Ia) or tangential (FCD Type Ib) dyslamination of the neocortex, microscopically identified in one or multiple lobes. FCD Type II is an isolated lesion characterized by cortical dyslamination and dysmorphic neurons without (Type IIa) or with balloon cells (Type IIb). Hence, the major change since a prior classification represents the introduction of FCD Type III, which occurs in combination with hippocampal sclerosis (FCD Type IIIa), or with epilepsy‐associated tumors (FCD Type IIIb). FCD Type IIIc is found adjacent to vascular malformations, whereas FCD Type IIId can be diagnosed in association with epileptogenic lesions acquired in early life (i.e., traumatic injury, ischemic injury or encephalitis). Significance: This three‐tiered classification system will be an important basis to evaluate imaging, electroclinical features, and postsurgical seizure control as well as to explore underlying molecular pathomechanisms in FCD.     

Epileptogenesis and reduced inward rectifier potassium current in tuberous sclerosis complex-1-deficient astrocytes

PURPOSE: Individuals with tuberous sclerosis complex (TSC) frequently have intractable epilepsy. To gain insights into mechanisms of epileptogenesis in TSC, we previously developed a mouse model of TSC with conditional inactivation of the Tsc1 gene in glia (Tsc1(GFAP)CKO mice). These mice develop progressive seizures, suggesting that glial dysfunction may be involved in epileptogenesis in TSC. Here, we investigated the hypothesis that impairment of potassium uptake through astrocyte inward rectifier potassium (Kir) channels may contribute to epileptogenesis in Tsc1(GFAP)CKO mice. METHODS: Kir channel function and expression were examined in cultured Tsc1-deficient astrocytes. Kir mRNA expression was analyzed in astrocytes microdissected from neocortical sections of Tsc1(GFAP)CKO mice. Physiological assays of astrocyte Kir currents and susceptibility to epileptiform activity induced by increased extracellular potassium were further studied in situ in hippocampal slices. RESULTS: Cultured Tsc1-deficient astrocytes exhibited reduced Kir currents and decreased expression of specific Kir channel protein subunits, Kir2.1 and Kir6.1. mRNA expression of the same Kir subunits also was reduced in astrocytes from neocortex of Tsc1(GFAP)CKO mice. By using pharmacologic modulators of signalling pathways implicated in TSC, we showed that the impairment in Kir channel function was not affected by rapamycin inhibition of the mTOR/S6K pathway, but was reversed by decreasing CDK2 activity with roscovitine or retinoic acid. Last, hippocampal slices from Tsc1(GFAP)CKO mice exhibited decreased astrocytic Kir currents, as well as increased susceptibility to potassium-induced epileptiform activity. CONCLUSIONS: Impaired extracellular potassium uptake by astrocytes through Kir channels may contribute to neuronal hyperexcitability and epileptogenesis in a mouse model of TSC.     

难治性颞叶癫癎患者脑内KCNJ4基因表达的研究

目的观察内向整流钾离子通道亚单位基因KCNJ4 mRNA及其编码的蛋白产物Kir2.3在难治性颞叶癫癎患者和急性脑外伤患者脑内的表达差异,从分子水平探讨难治性颞叶癫癎可能的发病机制.方法 12例难治性颞叶癫癎患者手术切除的颞叶组织,10例急性脑外伤患者相应部位颞叶组织,利用逆转录-聚合酶链反应(RT-PCR)与蛋白印迹检测(Western-blot)方法检测两组间KCNJ4 mRNA(KCNJ4 mRNA/β-actin)与Kir2.3通道蛋白(Kir2.3/β-actin)的表达.结果难治性颞叶癫癎患者与脑外伤患者比较,颞叶组织KCNJ4 mRNA(0.438±0.178)及Kir2.3通道蛋白(0.063)的表达水平均降低,差异有统计学意义(P＜0.05).结论 KCNJ4 mRNA表达水平的下调及其编码产物Kir2.3通道蛋白表达水平的下降,可能是难治性颞叶癫癎发生发展的重要的分子学基础之一.     

NPY gene transfer in the hippocampus attenuates synaptic plasticity and learning

Recombinant adeno-associated viral (rAAV) vector-induced neuropeptide Y (NPY) overexpression in the hippocampus exerts powerful antiepileptic and antiepileptogenic effects in rats. Such gene therapy approach could be a valuable alternative for developing new antiepileptic treatment strategies. Future clinical progress, however, requires more detailed evaluation of possible side effects of this treatment. Until now it has been unknown whether rAAV vector-based NPY overexpression in the hippocampus alters normal synaptic transmission and plasticity, which could disturb learning and memory processing. Here we show, by electrophysiological recordings in CA1 of the hippocampal formation of rats, that hippocampal NPY gene transfer into the intact brain does not affect basal synaptic transmission, but slightly alters short-term synaptic plasticity, most likely via NPY Y2 receptor-mediated mechanisms. In addition, transgene NPY seems to be released during high frequency neuronal activity, leading to decreased glutamate release in excitatory synapses. Importantly, memory consolidation appears to be affected by the treatment. We found that long-term potentiation (LTP) in the CA1 area is partially impaired and animals have a slower rate of hippocampal-based spatial discrimination learning. These data provide the first evidence that rAAV-based gene therapy using NPY exerts relative limited effect on synaptic plasticity and learning in the hippocampus, and therefore this approach could be considered as a viable alternative for epilepsy treatment.     

Kainic acid-induced sprouting of dynorphin- and enkephalin- containing mossy fibers in the dentate gyrus of the rat hippocampus

This study utilized Timm histochemistry and immunocytochemistry to determine the prolonged effects of kainic acid on the distribution of dynorphin- and enkephalin-containing mossy fibers in the rat dentate gyrus at progressive time points following kainic acid injection. Beginning 1–2 weeks after kainic acid administration, a progressive increase in the distribution and intensity of staining for supragranular zinc, dynorphin and enkephalin was observed in the dentate gyrus. The kainic acid-induced sprouting of mossy fibers containing dynorphin and enkephalin strongly resembles the pattern observed in the dentate gyrus of humans with temporal lobe epilepsy.     

Hemicranial Volume Deficits in Patients with Temporal Lobe Epilepsy With and Without Hippocampal Sclerosis

Summary: Purpose: In patients with refractory temporal lobe epilepsy, studies have suggested volume deficits measured by MRI of brain structures outside the epileptogenic hippocampus. Hippocampal sclerosis (HS) is a frequent, but not obligate, finding in such patients. The present study examines the influence of the presence of HS on quantitative magnetic resonance imaging (MRI) measurements.
Methods: We analyzed 47 patients and 30 controls by quantitative MRI, including intracranial volume (ICV), hemicranial volume, hippocampal volume (HCV), and T₂relaxometry. MRI results were compared with histological findings in the resected temporal lobe.
Results: Histology documented HS in 35 patients (HS group) and other findings in 12 patients (no-HS group). In both groups, the hemicranial volume ipsilateral to the epileptogenic focus was significantly smaller than on the contralateral side (p <0.004). The HCV on both sides was smaller in the HS group compared with patients without HS (p ≥ 0.004). Unilateral hippocampal atrophy and increased T, value were found in 71% of patients with HS, and bilaterally normal HCV and T, value were found in 67% of patients without HS.
Conclusions: The smaller hemicranial volume on the focus side, irrespective of the presence or absence of HS suggests a different pathogenic mechanism for the additional hemicranial volume deficit, compared to HS itself. The contralateral HCV deficit depends on the presence of HS, indicating a pathogenic connection between damage to both hippocampi.     

Changes in chloride homeostasis-regulating gene expressions in the rat hippocampus following amygdala kindling

In a rat kindling model, we examined expression patterns of NKCC1, KCC1, KCC2, and CLC-2. In the dentate granule cell layer, there was an activity-dependent increase in NKCC1 mRNA but significant decreases in KCC1 and CLC-2 mRNAs. In addition, CLC-2 mRNA expression was markedly decreased in CA1 pyramidal layer. These results suggest that an increase in [Cl⁻]_i and a resultant reduction in GABAergic inhibition may occur in hippocampus of epileptic rats.     

Cation-chloride cotransporters and GABA-ergic innervation in the human epileptic hippocampus

Intracellular chloride concentration, [Cl(-)](i), determines the polarity of GABA(A)-induced neuronal Cl(-) currents. In neurons, [Cl(-)](i) is set by the activity of Na(+), K(+), 2Cl(-) cotransporters (NKCC) such as NKCC1, which physiologically accumulate Cl(-) in the cell, and Cl(-) extruding K(+), Cl(-) cotransporters like KCC2. Alterations in the balance of NKCC1 and KCC2 activity may determine the switch from hyperpolarizing to depolarizing effects of GABA, reported in the subiculum of epileptic patients with hippocampal sclerosis. We studied the expression of NKCC (putative NKCC1) and KCC2 in human normal temporal neocortex by Western blot analysis and in normal and epileptic regions of the subiculum and the hippocampus proper using immunocytochemistry. Western blot analysis revealed NKCC and KCC2 proteins in adult human neocortical membranes similar to those in rat neocortex. NKCC and KCC2 immunolabeling of pyramidal and nonpyramidal cells was found in normal and epileptic hippocampal formation. In the transition between the subiculum with sclerotic regions of CA1, known to exhibit epileptogenic activity, double immunolabeling of NKCC and KCC2 revealed that approximately 20% of the NKCC-immunoreactive neurons do not express KCC2. In these same areas some neurons were distinctly hyperinnervated by parvalbumin (PV) positive hypertrophic basket formations that innervated mostly neurons expressing NKCC (74%) and to a lesser extent NKCC-immunonegative neurons (26%). Hypertrophic basket formations also innervated KCC2-positive (76%) and -negative (24%) neurons. The data suggest that changes in the relative expression of NKCC1 and KCC2 in neurons having aberrant GABA-ergic hyperinnervation may contribute to epileptiform activity in the subicular regions adjacent to sclerotic areas of the hippocampus.     

颞叶癫痫患者海马硬化神经元脱失的亚群特点

目的探讨颞叶癫痫患者海马硬化神经元脱失的亚群特点。方法颞叶癫痫患者中8例海马硬化和7例非海马硬化，以正常海马解剖为对照，观察两患者组海马各亚群神经元脱失情况。结果海马硬化组和非海马硬化组均有CA1、CA3锥体细胞和颗粒细胞脱失，海马硬化组明显。门区神经元脱失仅在海马硬化组可见。结论颞叶癫痫患者海马结构主要细胞脱失，门区神经元脱失是海马硬化的主要特点。海马硬化的形成与齿状回尤其是门区神经元的关系更为密切。     

GABAA α2 mRNA levels are decreased following induction of spontaneous epileptiform discharges in hippocampal-entorhinal cortical slices

Exposure of hippocampal slices to Mg²⁺ free media (0 Mg) has been shown to trigger full production of stimulus-induced seizure activity after restoration of physiological conditions [1]. In the present study employing hippocampal entorhinal cortical slices (HEC), spontaneous epileptiform discharges (SEDs) were induced using 0 Mg treatment following the return of the slices to physiological conditions. To evaluate the effect of sustained epileptiform activity on gene expression in this HEC slice preparation, changes in mRNA levels of the GABA_A α1 and α2 and β CaM Kinase II subunits were measured using in situ hybridization. HEC slices were incubated in oxygenated artificial cerebrospinal fluid (ACSF) in the presence or absence of Mg²⁺ for 3 h, then placed in oxygenated ACSF containing Mg²⁺ for up to 3 h. Control slices were maintained in Mg²⁺ containing ACSF for up to 6 h. Recurrent SEDs were observed in 0 Mg pre-treated slices while no epileptiform discharges were seen in control slices. Following induction of SEDs by 0 Mg pre-treatment, a significant decrease in mRNA encoding GABA_A α2 was found in the CA1, CA2, CA3 and dentate gyrus (DG) regions of the hippocampus for up to 3 h after treatment. Levels of mRNA for GABA_A α1 and β CaM Kinase II were not affected. The results document a decrease in GABA_A α2 gene expression following the induction of SEDs in the HEC slice preparation and suggest that rapid changes in neuronal gene expression may contribute to long lasting excitability changes associated with the induction of epilepsy.     

Hippocampal NPY gene transfer attenuates seizures without affecting epilepsy-induced impairment of LTP

Recently, hippocampal neuropeptide Y (NPY) gene therapy has been shown to effectively suppress both acute and chronic seizures in animal model of epilepsy, thus representing a promising novel antiepileptic treatment strategy, particularly for patients with intractable mesial temporal lobe epilepsy (TLE). However, our previous studies show that recombinant adeno-associated viral (rAAV)-NPY treatment in naive rats attenuates long-term potentiation (LTP) and transiently impairs hippocampal learning process, indicating that negative effect on memory function could be a potential side effect of NPY gene therapy.Here we report how rAAV vector-mediated overexpression of NPY in the hippocampus affects rapid kindling, and subsequently explore how synaptic plasticity and transmission is affected by kindling and NPY overexpression by field recordings in CA1 stratum radiatum of brain slices. In animals injected with rAAV-NPY, we show that rapid kindling-induced hippocampal seizures in vivo are effectively suppressed as compared to rAAV-empty injected (control) rats. Six to nine weeks later, basal synaptic transmission and short-term synaptic plasticity are unchanged after rapid kindling, while LTP is significantly attenuated in vitro. Importantly, transgene NPY overexpression has no effect on short-term synaptic plasticity, and does not further compromise LTP in kindled animals. These data suggest that epileptic seizure-induced impairment of memory function in the hippocampus may not be further affected by rAAV-NPY treatment, and may be considered less critical for clinical application in epilepsy patients already experiencing memory disturbances.     

Variable epilepsy phenotypes associated with a familial intragenic deletion of the SCN1A gene

Deletions and duplications/amplifications of the α1-sodium channel subunit (SCN1A) gene occur in about 12% of patients with Dravet syndrome (DS) who are otherwise mutation-negative. Such genomic abnormalities cause loss of function, with severe phenotypes, reproductive disadvantage and, therefore, sporadic occurrence. Inherited mutations, occurring in ～5% of patients with DS, are usually missense; transmission occurs from a mildly affected parent exhibiting febrile seizures (FS) or the generalized epilepsy with febrile seizures plus (GEFS+) spectrum. We identified an intragenic SCN1A deletion in a three-generation, clinically heterogeneous family. Sequence analysis of SCN9A, a putative modifier, ruled out pathogenic mutations, variants, or putative disease-associated haplotype segregating with phenotype severity. Intrafamilial variability in phenotype severity indicates that SCN1A loss of function causes a phenotypic spectrum in which seizures precipitated by fever are prominent and schematic syndrome subdivisions would be inappropriate. SCN1A deletions should be ruled out even in individuals with mild phenotypes.