Evaluation of HCN2 abnormalities as a cause of juvenile audiogenic seizures in Black Swiss mice

Epilepsy is an often-debilitating disease with many etiologies. Genetic predisposition is common for many of the generalized epilepsy syndromes, and mutations in genes encoding neuronal ion channels are causative in many cases. We previously identified a locus for juvenile audiogenic monogenic seizures (jams1) in the Black Swiss mouse strain, delimited by the gene basigin (Bsg) and the marker D10Mit140. This region includes Hcn2, the gene encoding the hyperpolarization-activated cyclic nucleotide-gated channel subunit 2 (HCN2), an ion channel implicated in epilepsy. By sequencing genomic DNA, we found that Black Swiss mice have a single polymorphism in exon 2 within the Hcn2 gene. This single G/C to A/T base change alters the third position of a codon specifying alanine residue 293, without changing the predicted amino acid sequence. Furthermore, we found no detectable differences in HCN2 protein expression in the brains of Black Swiss mice, compared to control mice. We therefore reason that juvenile audiogenic seizures in Black Swiss mice are unlikely to be due to abnormalities of HCN2 channel function.     

Efficacy of antiseizure medication in a mouse model of HCN1 developmental and epileptic encephalopathy

Acquisition of drug-sensitivity profiles is challenging in rare epilepsies. Anecdotal evidence suggests that antiseizure medications that block sodium channels as their primary mechanism of action exacerbate seizures in HCN1 developmental and epileptic encephalopathies (DEEs), whereas sodium valproate is effective for some patients. The Hcn1 M294L heterozygous knock-in (Hcn1^M294L) mouse carries the homologue of the recurrent gain-of-function HCN1 M305L pathogenic variant and recapitulates the seizure and some behavioral phenotypes observed in patients. We used this mouse model to study drug efficacy in HCN1 DEE. Hcn1^M294L mice display epileptiform spiking on electrocorticography (ECoG), which we used as a quantifiable measure of drug effect. Phenytoin, lamotrigine, and retigabine significantly increased ECoG spike frequency, with lamotrigine and retigabine triggering seizures in a subset of the mice tested. In addition, there was a strong trend for carbamazepine to increase spiking. In contrast, levetiracetam, diazepam, sodium valproate, and ethosuximide all significantly reduced ECoG spike frequency. Drugs that reduced spiking did not cause any consistent ECoG spectral changes, whereas drugs that increased spiking all increased power in the slower delta and/or theta bands. These data provide a framework on which to build our understanding of gain-of-function HCN1 DEE pharmacosensitivity in the clinical setting.     

Recessive loss-of-function mutation in the pacemaker HCN2 channel causing increased neuronal excitability in a patient with idiopathic generalized epilepsy

The hyperpolarization-activated I(h) current, coded for by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, controls synaptic integration and intrinsic excitability in many brain areas. Because of their role in pacemaker function, defective HCN channels are natural candidates for contributing to epileptogenesis. Indeed, I(h) is pathologically altered after experimentally induced seizures, and several independent data indicate a link between dysfunctional HCN channels and different forms of epilepsy. However, direct evidence for functional changes of defective HCN channels correlating with the disease in human patients is still elusive. By screening families with epilepsy for mutations in Hcn1 and Hcn2 genes, we found a recessive loss-of-function point mutation in the gene coding for the HCN2 channel in a patient with sporadic idiopathic generalized epilepsy. Of 17 screened members of the same family, the proband was the only one affected and homozygous for the mutation. The mutation (E515K) is located in the C-linker, a region known to affect channel gating. Functional analysis revealed that homomeric mutant, but not heteromeric wild-type/mutant channels, have a strongly inhibited function caused by a large negative shift of activation range and slowed activation kinetics, effectively abolishing the HCN2 contribution to activity. After transfection into acutely isolated newborn rat cortical neurons, homomeric mutant, but not heteromeric wild type/mutant channels, lowered the threshold of action potential firing and strongly increased cell excitability and firing frequency when compared with wild-type channels. This is the first evidence in humans for a single-point, homozygous loss-of-function mutation in HCN2 potentially associated with generalized epilepsy with recessive inheritance.     

h channel-dependent deficit of theta oscillation resonance and phase shift in temporal lobe epilepsy

I_h tunes hippocampal CA1 pyramidal cell dendrites to optimally respond to theta inputs (4–12 Hz), and provides a negative time delay to theta inputs. Decreased I_h activity, as seen in experimental temporal lobe epilepsy (TLE), could significantly alter the response of dendrites to theta inputs. Here we report a progressive erosion of theta resonance and phase lead in pyramidal cell dendrites during epileptogenesis in a rat model of TLE. These alterations were due to decreased I_h availability, via a decline in HCN1/HCN2 subunit expression resulting in decreased h currents, and altered kinetics of the residual channels. This acquired HCN channelopathy thus compromises temporal coding and tuning to theta inputs in pyramidal cell dendrites. Decreased theta resonance in vitro also correlated with a reduction in theta frequency and power in vivo. We suggest that the neuronal/circuitry changes associated with TLE, including altered I_h-dependent inductive mechanisms, can disrupt hippocampal theta function.     

Circadian distribution of generalized tonic-clonic seizures associated with murine succinic semialdehyde dehydrogenase deficiency, a disorder of GABA metabolism

Human succinic semialdehyde dehydrogenase (SSADH) deficiency is an autosomal recessive disorder of GABA metabolism associated with motor impairment and epileptic seizures. Similarly, mice with targeted deletion of the Aldh5a1 gene (Aldh5a1^–/–) exhibit SSADH deficiency and seizures early in life. These seizures begin as absence seizures the second week of life, but evolve into generalized convulsive seizures that increase in severity and become lethal during the fourth postnatal week. The seizures are alleviated and survival is prolonged when the mutant animals are weaned onto a ketogenic diet (KD). The persistence of spontaneous, recurrent, generalized tonic–clonic seizures in KD-treated adult Aldh5a1^–/– mice allowed us to quantify their daily (circadian) distribution using a novel behavioral method based on the detection of changes in movement velocity. Adult KD-treated Aldh5a1^–/– mice exhibited a seizure phenotype characterized by fits of wild running clonus accompanied by jumping and bouncing. These hypermotor seizures were largely spontaneous and occurred daily in a nonrandom pattern. The seizure rhythm showed a peak shortly after dark phase onset (2008 hours) with near-24-hour periodicity. Age-matched wild-type littermates showed no evidence of abnormal motor behavior. These new data suggest that generalized tonic–clonic seizures in Aldh5a1^–/– mice are more frequent during a specific time of day and will provide useful information to clinicians for the treatment of seizures associated with human SSADH deficiency.     

Increased number of GABAB receptors in the lethargic (lh/lh) mouse model of absence epilepsy

This study begins to explore mechanisms underlying the role of GABA_B receptors in absence seizures in lethargic (lh/lh) mice. To test the hypothesis that alterations intrinsic to the GABA_B receptor underlie enhanced synaptic activation of these receptors in absence seizures, we measured GABA-displaceable [³H]baclofen binding to neocortical plasma membranes prepared from lh/lh and wild (+/+) age-matched congenic mice. The number (B_max) of binding sites was significantly greater (20%) in lh/lh (4.2pmol/mg protein, n = 43 pairs, P < 0.02) than in +/+ mice (3.3 pmol/mg protein) in an age-dependent manner. Interestingly, the subject of lh/lh mice with greater seizure frequency (40–70 seizures/15 min, measured by bipolar electrodes implanted into neocortex; n = 11) had a significantly greater B_max (P < 0.003) than the subset with lower seizure frequency (1–20 seizures/15 min; n = 11). The equilibrium dissociation constant (K_d) was unchanged (60 nM in both). The K_d of both strains was inhibited to an equal degree by the nonhydrolysable GTP analogue 5′-guanylimido-diphosphate [Gpp(NH)p]. The increased number of GABA_B binding sites was selective, because binding to NMDA sites ([³H]glutamate binding) and to GABA_A sites ([³H]muscimol binding) was not significantly different in the two strains. These data suggest that the increased number of GABA_B receptors in lh/lh mice underlies enhanced synaptic activation of these receptors. Together with evidence that GABA_B receptor activation can produce disinhibition, our data support a role for GABA_B receptors in the expression of absence seizures in lh/lh mice.     

Altered thalamocortical rhythmicity in Ca(v)2.3-deficient mice

Voltage-gated calcium channels (VGCCs) are key regulators of neuronal excitability and important factors in epileptogenesis and neurodegeneration. Recent findings suggest a novel, important proictogenic and proneuroapoptotic role of the Ca_v2.3 E/R-type VGCCs in convulsive generalized tonic–clonic and hippocampal seizures. Though Ca_v2.3 is also expressed in key structures of the thalamocortical circuitry, their functional relevance in non-convulsive absence seizure activity remains unknown. To this end, we investigated absence specific spike–wave discharge (SWD) susceptibility in control and Ca_v2.3-deficient mice by systemic administration of γ-hydroxybutyrolactone (GBL, 70 mg/kg i.p.), followed by electrocorticographic radiotelemetric recordings, behavioral analysis and histomorphological characterization. Based on motoric studies, SWD and power-spectrum density (PSD) analysis, our results demonstrate that Ca_v2.3^−/− mice exhibit increased absence seizure susceptibility and altered absence seizure architecture compared to control animals. This study provides evidence for the first time that Ca_v2.3 E/R-type Ca²⁺ channels are important in modulating thalamocortical hyperoscillation exerting anti-epileptogenic effects in non-convulsive absence seizures.     

Extended spectrum of idiopathic generalized epilepsies associated with CACNA1H functional variants

Objective

The relationship between genetic variation in the T‐type calcium channel gene CACNA1H and childhood absence epilepsy is well established. The purpose of this study was to investigate the range of epilepsy syndromes for which CACNA1H variants may contribute to the genetic susceptibility architecture and determine the electrophysiological effects of these variants in relation to proposed mechanisms underlying seizures.

Methods

Exons 3 to 35 of CACNA1H were screened for variants in 240 epilepsy patients (167 unrelated) and 95 control subjects by single‐stranded conformation analysis followed by direct sequencing. Cascade testing of families was done by sequencing or single‐stranded conformation analysis. Selected variants were introduced into the CACNA1H protein by site‐directed mutagenesis. Constructs were transiently transfected into human embryo kidney cells, and electrophysiological data were acquired.

Results

More than 100 variants were detected, including 19 novel variants leading to amino acid changes in subjects with phenotypes including childhood absence, juvenile absence, juvenile myoclonic and myoclonic astatic epilepsies, as well as febrile seizures and temporal lobe epilepsy. Electrophysiological analysis of 11 variants showed that 9 altered channel properties, generally in ways that would be predicted to increase calcium current.

Interpretation

Variants in CACNA1H that alter channel properties are present in patients with various generalized epilepsy syndromes. We propose that these variants contribute to an individual's susceptibility to epilepsy but are not sufficient to cause epilepsy on their own. The genetic architecture is dominated by rare functional variants; therefore, CACNA1H would not be easily identified as a susceptibility gene by a genome‐wide case–control study seeking a statistical association. Ann Neurol 2007     

Epilepsy in succinic semialdehyde dehydrogenase deficiency, a disorder of GABA metabolism

Objectives

Succinic semialdehyde dehydrogenase (SSADH) deficiency is a gamma-aminobutyric acid (GABA) degradative defect. Epilepsy affects half of patients. The murine model is associated with a transition from absence to convulsive seizures in the third week, with fatal status epilepticus.

Methods

The clinical phenotype is reported from a patient database. Flumazenil-Positron Emission Topography (FMZ-PET) and Transcranial Magnetic Stimulation (TMS) were used to study GABA neurotransmission. Electrocorticography, single cell electrophysiology, and radioligand binding studies are reported from animal studies.

Results

Generalized seizures predominate, including tonic–clonic, atypical absence, and myoclonic. EEG discharges are typically generalized spike-wave. MRI shows a dentatopallidoluysian pattern. Sudden Unexpected Death in Epilepsy Patients (SUDEP) has occurred and the associated neuropathology reveals chronic excitotoxic injury in gloubus pallidus. Investigations using FMZ-PET and TMS support downregulation of GABA_A and GABA_B activity, respectively, in patients. Gamma-hydroxybutyrate (GHB) induces spike-wave discharges in homozygous null mice via GHB and GABA_B-mediated mechanisms. These resemble absence seizures and are abolished by a GABA_B receptor antagonist. Decreased binding of GABA_A and GABA_B receptor antagonists has been demonstrated in P19 and P14 null mice, respectively. Downregulation of GABA_A and GABA_B receptor subunits is observed by P14. GABA_A and GABA_B mediated potentials are reduced from P8-P14.

Conclusion

Generalized epilepsy and epileptiform discharges are characteristic of SSADH deficiency. Spontaneous absence seizures appear in null mice by the third week, which may be induced by GHB or GABA_B activity. Subsequent overuse dependent downregulation of GABA_A and GABA_B receptor activity may be associated with hyperexcitability concomitant with the transition to generalized seizures.     

Increased seizure severity and seizure‐related death in mice lacking HCN1 channels

Persistent down‐regulation in the expression of the hyperpolarization‐activated HCN1 cation channel, a key determinant of intrinsic neuronal excitability, has been observed in febrile seizure, temporal lobe epilepsy, and generalized epilepsy animal models, as well as in patients with epilepsy. However, the role and importance of HCN1 down‐regulation for seizure activity is unclear. To address this question we determined the susceptibility of mice with either a general or forebrain‐restricted deletion of HCN1 to limbic seizure induction by amygdala kindling or pilocarpine administration. Loss of HCN1 expression in both mouse lines is associated with higher seizure severity and higher seizure‐related mortality, independent of the seizure‐induction method used. Therefore, down‐regulation of HCN1 associated with human epilepsy and rodent models may be a contributing factor in seizure behavior.     

HCN channels expressed in the inner ear are necessary for normal balance function

HCN1-4 subunits form Na+/K+-permeable ion channels that are activated by hyperpolarization and carry the current known as I(h). I(h) has been characterized in vestibular hair cells of the inner ear, but its molecular correlates and functional contributions have not been elucidated. We examined Hcn mRNA expression and immunolocalization of HCN protein in the mouse utricle, a mechanosensitive organ that contributes to the sense of balance. We found that HCN1 is the most highly expressed subunit, localized to the basolateral membranes of type I and type II hair cells. We characterized I(h) using the whole-cell, voltage-clamp technique and found the current expressed in 84% of the cells with a mean maximum conductance of 4.4 nS. I(h) was inhibited by ZD7288, cilobradine, and by adenoviral expression of a dominant-negative form of HCN2. To determine which HCN subunits carried I(h), we examined hair cells from mice deficient in Hcn1, 2, or both. I(h) was completely abolished in hair cells of Hcn1?/? mice and Hcn1/2?/? mice but was similar to wild-type in Hcn2?/? mice. To examine the functional contributions of I(h), we recorded hair cell membrane responses to small hyperpolarizing current steps and found that activation of I(h) evoked a 5-10 mV sag depolarization and a subsequent 15-20 mV rebound upon termination. The sag and rebound were nearly abolished in Hcn1-deficient hair cells. We also found that Hcn1-deficient mice had deficits in vestibular-evoked potentials and balance assays. We conclude that HCN1 contributes to vestibular hair cell function and the sense of balance.     

Proton magnetic resonance spectroscopic imaging for discrimination of absence and complex partial seizures

We performed proton magnetic resonance spectroscopic imaging of the temporal lobes between, during, and soon after nonconvulsive seizures in 20 patients with documented temporal lobe epilepsy, 5 patients with primary generalized epilepsy, and 2 patients with secondary generalized epilepsy. Our objective was to determine whether there were metabolic changes observable by magnetic resonance spectroscopic imaging during seizures and whether these changes were specific for focal or generalized nonconvulsive seizures. We found a significant increase in lactate to creatine plus phosphocreatine (lactate/creatine) values, reflecting an imbalance in energy supply and demand or an adaptation in response to ictal neuronal discharges, during and soon after complex partial seizures, but not during or soon after absence seizures associated with generalized epilepsy. In patients with temporal lobe epilepsy, the N-acetylaspartate resonance relative to creatine plus phosphocreatine was low in one or both temporal lobes, indicating neuronal loss or damage. This was not observed in patients with primary generalized epilepsy. The regions with abnormal lactate/creatine and N-acetylaspartate/creatine values corresponded to the epileptogenic focus as defined by clinical-electroencephalographic investigation. There was no change in the N-acetylaspartate/creatine values in the temporal lobes between the interictal, ictal, or postictal states. We conclude that (1) partial seizures are associated with abnormally high lactate levels, but absence seizures are not, and (2) no short-term changes of N-acetylaspartate occur during or soon after complex partial seizures or absence seizures. These findings may be related to the lack of postictal confusion in patients with absence seizures, as well as with the more benign course of primary generalized epilepsy with nonconvulsive attacks.     

Cacna1g is a genetic modifier of epilepsy in a mouse model of Dravet syndrome

Dravet syndrome, an early onset epileptic encephalopathy, is most often caused by de novo mutation of the neuronal voltage‐gated sodium channel gene SCN1A. Mouse models with deletion of Scn1a recapitulate Dravet syndrome phenotypes, including spontaneous generalized tonic–clonic seizures, susceptibility to seizures induced by elevated body temperature, and elevated risk of sudden unexpected death in epilepsy. Importantly, the epilepsy phenotype of Dravet mouse models is highly strain‐dependent, suggesting a strong influence of genetic modifiers. We previously identified Cacna1g, encoding the Cav3.1 subunit of the T‐type calcium channel family, as an epilepsy modifier in the Scn2a^Q54 transgenic epilepsy mouse model. In this study, we asked whether transgenic alteration of Cacna1g expression modifies severity of the Scn1a^+/? Dravet phenotype. Scn1a^+/? mice with decreased Cacna1g expression showed partial amelioration of disease phenotypes with improved survival and reduced spontaneous seizure frequency. However, reduced Cacna1g expression did not alter susceptibility to hyperthermia‐induced seizures. Transgenic elevation of Cacna1g expression had no effect on the Scn1a^+/? epilepsy phenotype. These results provide support for Cacna1g as a genetic modifier in a mouse model of Dravet syndrome and suggest that Cav3.1 may be a potential molecular target for therapeutic intervention in patients.     

Idiopathische generalisierte Epilepsien

Idiopathic epilepsies are genetically determined. The inheritance can be either monogenic, considering a single gene mutation as sufficient to cause the phenotype, or mainly complex, when the epileptic phenotype is determined by several genetic factors. The most important and most common subtypes of idiopathic generalized epilepsies (IGE) are childhood and juvenile absence epilepsies (CAE, JAE), juvenile myoclonic epilepsy (JME), and epilepsy with grand mal (generalized tonic-clonic) seizures on awakening (EGMA); closely related are early onset absence epilepsy (EOAE) and generalized/genetic epilepsy with febrile seizures plus (GEFS+). The IGE subtypes are characterized by primary generalized seizure types, an age-dependent onset, typical pathological EEG patterns, a benign course and normal psychomotor development. In recent years, an increasing number of mutations mainly associated with rare monogenic idiopathic epilepsy syndromes have been identified in genes encoding subunits of voltage- or ligand-gated ion channels, such as mutations in Na⁺ channel and GABA_(A) receptor subunits in GEFS+. A few mutations have also been detected in the common forms of IGE, mainly in GABA_(A) receptor subunits conferring a neuronal dysinhibition. For absence seizures, particularly EOAE, mutations in the glucose transporter type 1 (GLUT1) have been described, leading to a reduced transport rate of glucose across the blood-brain barrier. Recently, chromosomal microdeletions were found in up to 2.5% of IGE patients as a significant risk factor. Due to the complex genetic trait, the genetic enigma of IGE is just starting to be unraveled. The next generation sequencing techniques available now enable exome- and genome-wide sequence analyses, which the authors hope will contribute to understanding the pathophysiology of these common forms of epilepsy.     

Augmented currents of an HCN2 variant in patients with febrile seizure syndromes

The genetic architecture of common epilepsies is largely unknown. HCNs are excellent epilepsy candidate genes because of their fundamental neurophysiological roles. Screening in subjects with febrile seizures and genetic epilepsy with febrile seizures plus revealed that 2.4% carried a common triple proline deletion (delPPP) in HCN2 that was seen in only 0.2% of blood bank controls. Currents generated by mutant HCN2 channels were ∼35% larger than those of controls; an effect revealed using automated electrophysiology and an appropriately powered sample size. This is the first association of HCN2 and familial epilepsy, demonstrating gain of function of HCN2 current as a potential contributor to polygenic epilepsy. ANN NEUROL 2010;67:542–546     

Loss of β1 accessory Na+ channel subunits causes failure of carbamazepine,but not of lacosamide,in blocking high‐frequency firing via differential effects on persistent Na+ currents

Purpose: In chronic epilepsy, a substantial proportion of up to 30% of patients remain refractory to antiepileptic drugs (AEDs). An understanding of the mechanisms of pharmacoresistance requires precise knowledge of how AEDs interact with their targets. Many commonly used AEDs act on the transient and/or the persistent components of the voltage‐gated Na⁺ current (I_NaT and I_NaP, respectively). Lacosamide (LCM) is a novel AED with a unique mode of action in that it selectively enhances slow inactivation of fast transient Na⁺ channels. Given that functional loss of accessory Na⁺ channel subunits is a feature of a number of neurologic disorders, including epilepsy, we examined the effects of LCM versus carbamazepine (CBZ) on the persistent Na⁺ current (I_NaP), in the presence and absence of accessory subunits within the channel complex. Methods: Using patch‐clamp recordings in intact hippocampal CA1 neurons of Scn1b null mice, I_NaP was recorded using slow voltage ramps. Application of 100 μm CBZ or 300 μm LCM reduced the maximal I_NaP conductance in both wild‐type and control mice. Key Findings: As shown previously by our group in Scn1b null mice, CBZ induced a paradoxical increase of I_NaP conductance in the subthreshold voltage range, resulting in an ineffective block of repetitive firing in Scn1b null neurons. In contrast, LCM did not exhibit such a paradoxical increase, and accordingly maintained efficacy in blocking repetitive firing in Scn1b null mice. Significance: These results suggest that the novel anticonvulsant LCM maintains activity in the presence of impaired Na⁺ channel β₁ subunit expression and thus may offer an improved efficacy profile compared with CBZ in diseases associated with an impaired expression of β sub‐units as observed in epilepsy.