Age-Dependent Activation of Purinergic Transmission Contributes to the Development of Epileptogenesis in ADSHE Model Rats.

To explore the developmental processes of epileptogenesis/ictogenesis, this study determined age-dependent functional abnormalities associated with purinergic transmission in a genetic rat model (S286L-TG) of autosomal-dominant sleep-related hypermotor epilepsy (ADSHE). The age-dependent fluctuations in the release of ATP and L-glutamate in the orbitofrontal cortex (OFC) were determined using microdialysis and ultra-high-performance liquid chromatography with mass spectrometry (UHPLC-MS). ATP release from cultured astrocytes was also determined using UHPLC-MS. The expressions of P2X7 receptor (P2X7R), connexin 43, phosphorylated-Akt and phosphorylated-Erk were determined using capillary immunoblotting. No functional abnormalities associated with purinergic transmission could be detected in the OFC of 4-week-old S286L-TG and cultured S286L-TG astrocytes. However, P2X7R expression, as well as basal and P2X7R agonist-induced ATP releases, was enhanced in S286L-TG OFC in the critical ADSHE seizure onset period (7-week-old). Long-term exposure to a modest level of P2X7R agonist, which could not increase astroglial ATP release, for 14 d increased the expressions of P2X7R and connexin 43 and the signaling of Akt and Erk in astrocytes, and it enhanced the sensitivity of P2X7R to its agonists. Akt but not Erk increased P2X7R expression, whereas both Akt and Erk increased connexin 43 expression. Functional abnormalities, enhanced ATP release and P2X7R expression were already seen before the onset of ADSHE seizure in S286L-TG. Additionally, long-term exposure to the P2X7R agonist mimicked the functional abnormalities associated with purinergic transmission in astrocytes, similar to those in S286L-TG OFC. Therefore, these results suggest that long-term modestly enhanced purinergic transmission and/or activated P2X7R are, at least partially, involved in the development of the epileptogenesis of ADSHE, rather than that of ictogenesis.

Age-Dependent Activation of Pannexin1 Function Contributes to the Development of Epileptogenesis in Autosomal Dominant Sleep-related Hypermotor Epilepsy Model Rats.

To explore the processes of epileptogenesis/ictogenesis, this study determined the age-dependent development of the functional abnormalities in astroglial transmission associated with pannexin1-hemichannel using a genetic rat model of autosomal dominant sleep-related hypermotor epilepsy (ADSHE) named 'S286L-TG'. Pannexin1 expression in the plasma membrane of primary cultured cortical astrocytes and the orbitofrontal cortex (OFC), which is an ADSHE focus region, were determined using capillary immunoblotting. Astroglial D-serine releases induced by artificial high-frequency oscillation (HFO)-evoked stimulation, the removal of extracellular Ca2+, and the P2X7 receptor agonist (BzATP) were determined using ultra-high performance liquid chromatography (UHPLC). The expressions of pannexin1 in the plasma membrane fraction of the OFC in S286L-TG at four weeks old were almost equivalent when compared to the wild type. The pannexin1 expression in the OFC of the wild type non-statistically decreased age-dependently, whereas that in S286L-TG significantly increased age-dependently, resulting in relatively increasing pannexin1 expression from the 7- (at the onset of interictal discharge) and 10-week-old (after the ADSHE seizure onset) S286L-TG compared to the wild type. However, no functional abnormalities of astroglial pannexin1 expression or D-serine release through the pannexin1-hemichannels from the cultured astrocytes of S286L-TG could be detected. Acutely HFO-evoked stimulation, such as physiological ripple burst (200 Hz) and epileptogenic fast ripple burst (500 Hz), frequency-dependently increased both pannexin1 expression in the astroglial plasma membrane and astroglial D-serine release. Neither the selective inhibitors of pannexin1-hemichannel (10PANX) nor connexin43-hemichannel (Gap19) affected astroglial D-serine release during the resting stage, whereas HFO-evoked D-serine release was suppressed by both inhibitors. The inhibitory effect of 10PANX on the ripple burst-evoked D-serine release was more predominant than that of Gap19, whereas fast ripple burst-evoked D-serine release was predominantly suppressed by Gap19 rather than 10PANX. Astroglial D-serine release induced by acute exposure to BzATP was suppressed by 10PANX but not by Gap19. These results suggest that physiological ripple burst during the sleep spindle plays important roles in the organization of some components of cognition in healthy individuals, but conversely, it contributes to the initial development of epileptogenesis/ictogenesis in individuals who have ADSHE vulnerability via activation of the astroglial excitatory transmission associated with pannexin1-hemichannels.

Exome Sequencing in an ADSHE Family: VUS Identification and Limits.

Autosomal dominant sleep-related hypermotor epilepsy (ADSHE) is the familial form of a focal epilepsy characterized by hyperkinetic focal seizures, mainly arising during non-rapid eye movements (NREM) sleep. Mutations associated with ADSHE account for a small proportion of the genetically determined cases, suggesting the existence of other disease-causing genes. Here, we reported the results obtained by performing trio-based whole-exome sequencing (WES) in an Italian family showing ADSHE and investigated the structural impact of putative variants by in silico modeling analysis. We identified a p.(Trp276Gly) variant in MOXD1 gene encoding the monooxigenase DBH like 1 protein, cosegregating with the disease and annotated as VUS under the ACMG recommendations. Structural bioinformatic analysis predicted a high destabilizing effect of this variant, due to the loss of important hydrophilic bonds and an expansion of cavity volume in the protein hydrophobic core. Although our data support a functional effect of the p.(Trp276Gly) variant, we highlight the need to identify additional families carrying MOXD1 mutations or functional analyses in suitable models to clarify its role in ADSHE pathogenesis. Moreover, we discuss the importance of VUS reporting due to the low rate of pathogenic variant identification by NGS in epilepsy and for future reinterpretation studies.

Autosomal dominant sleep-related hypermotor epilepsy caused by a previously unreported CHRNA4 variant.

Autosomal dominant sleep-related hypermotor epilepsy (ADSHE) is a rare heritable form of epilepsy. It is characterized by hypermotor seizures occurring mainly during sleep. Seizures are typically abrupt in onset and offset and tend to increase in complexity and duration during the night. ADSHE is inherited in an autosomal dominant manner, and penetrance is estimated to be 70%. We describe two brothers with ADSHE with a previously unreported variant in CHRNA4, and the effect of medical treatment with carbamazepine. We highlight the relevance of genetic testing in patients with atypical and clustering episodes of nightmares, night terrors, or panic attacks, as these patients could be misdiagnosed, and instead be suffering from ADSHE, a potentially treatable condition.

Targeting KNa1.1 channels in KCNT1-associated epilepsy.

Gain-of-function (GOF) pathogenic variants of KCNT1, the gene encoding the largest known potassium channel subunit, KNa1.1, are associated with developmental and epileptic encephalopathies accompanied by severe psychomotor and intellectual disabilities. Blocking hyperexcitable KNa1.1 channels with quinidine, a class I antiarrhythmic drug, has shown variable success in patients in part because of dose-limiting off-target effects, poor blood-brain barrier (BBB) penetration, and low potency. In recent years, high-resolution cryogenic electron microscopy (cryo-EM) structures of the chicken KNa1.1 channel in different activation states have been determined, and animal models of the diseases have been generated. Alongside increasing information about the functional effects of GOF pathogenic variants on KNa1.1 channel behaviour and how they lead to hyperexcitability, these tools will facilitate the development of more effective treatment strategies. We review the range of KCNT1 variants and their functional effects, the challenges posed by current treatment strategies, and recent advances in finding more potent and selective therapeutic interventions for KCNT1-related epilepsies.

Nicotinic Receptors in Sleep-Related Hypermotor Epilepsy: Pathophysiology and Pharmacology.

Sleep-related hypermotor epilepsy (SHE) is characterized by hyperkinetic focal seizures, mainly arising in the neocortex during non-rapid eye movements (NREM) sleep. The familial form is autosomal dominant SHE (ADSHE), which can be caused by mutations in genes encoding subunits of the neuronal nicotinic acetylcholine receptor (nAChR), Na+-gated K+ channels, as well as non-channel signaling proteins, such as components of the gap activity toward rags 1 (GATOR1) macromolecular complex. The causative genes may have different roles in developing and mature brains. Under this respect, nicotinic receptors are paradigmatic, as different pathophysiological roles are exerted by distinct nAChR subunits in adult and developing brains. The widest evidence concerns α4 and ß2 subunits. These participate in heteromeric nAChRs that are major modulators of excitability in mature neocortical circuits as well as regulate postnatal synaptogenesis. However, growing evidence implicates mutant α2 subunits in ADSHE, which poses interpretive difficulties as very little is known about the function of α2-containing (α2*) nAChRs in the human brain. Planning rational therapy must consider that pharmacological treatment could have different effects on synaptic maturation and adult excitability. We discuss recent attempts towards precision medicine in the mature brain and possible approaches to target developmental stages. These issues have general relevance in epilepsy treatment, as the pathogenesis of genetic epilepsies is increasingly recognized to involve developmental alterations.

Pathomechanism of nocturnal paroxysmal dystonia in autosomal dominant sleep-related hypermotor epilepsy with S284L-mutant α4 subunit of nicotinic ACh receptor.

To study the pathomechanism and pathophysiology of nocturnal paroxysmal dystonia of autosomal dominant sleep-related hypermotor epilepsy (ADSHE), this study determined functional abnormalities in thalamic hyperdirect pathway from reticular thalamic nucleus (RTN), motor thalamic nuclei (MoTN), subthalamic nucleus (STN) to substantia nigra pars reticulata (SNr) of transgenic rats (S286L-TG) bearing S286 L missense mutation of rat Chrna4 gene, which corresponds to the S284 L mutation in the human CHRNA4 gene. The activation of α4ß2-nAChR in the RTN increased GABA release in MoTN resulting in reduced glutamatergic transmission in thalamic hyperdirect pathway of wild-type. Contrary to wild-type, activation of S286L-mutant α4ß2-nAChR (loss-of-function) in the RTN relatively enhanced glutamatergic transmission in thalamic hyperdirect pathway of S286L-TG via impaired GABAergic inhibition in intra-thalamic (RTN-MoTN) pathway. These functional abnormalities in glutamatergic transmission in hyperdirect pathway contribute to the pathomechanism of electrophysiologically negative nocturnal paroxysmal dystonia of S286L-TG. Therapeutic-relevant concentration of zonisamide (ZNS) inhibited the glutamatergic transmission in the hyperdirect pathway via activation of group II metabotropic glutamate receptor (II-mGluR) in MoTN and STN. The present results suggest that S286L-mutant α4ß2-nAChR induces GABAergic disinhibition in intra-thalamic (RTN-MoTN) pathway and hyperactivation of glutamatergic transmission in thalamic hyperdirect pathway (MoTN-STN-SNr), possibly contributing to the pathomechanism of nocturnal paroxysmal dystonia of ADSHE patients with S284L mutant CHRNA4. Inhibition of glutamatergic transmission in thalamic hyperdirect pathway induced by ZNS via activation of II-mGluR may be involved, at least partially, in ZNS-sensitive nocturnal paroxysmal dystonia of ADSHE patients with S284L mutation.

Remarkable effect of transdermal nicotine in children with CHRNA4-related autosomal dominant sleep-related hypermotor epilepsy.

OBJECTIVE: Autosomal dominant sleep-related hypermotor epilepsy (ADSHE) is characterized by hypermotor seizures and may be caused by gain-of-function mutations affecting the nicotinic acetylcholine receptor (nAChR). Benefit from nicotine consumption has been reported in adult patients with this disorder. For the first time, the effect of transdermal nicotine is evaluated in children. METHODS: Transdermal nicotine was applied to three boys, two aged 10 years (7 mg/24 h) and one six years (3.5 mg/24 h). Autosomal dominant sleep-related hypermotor epilepsy was caused by the p.S280F-CHRNA4 (cholinergic receptor, nicotinic, alpha polypeptide 4) mutation. The children suffered from frequent, persistent nocturnal seizures and had developed educational and psychosocial problems. Seizure frequency and cognitive and behavioral parameters were assessed before and after treatment. RESULTS: A striking seizure reduction was reported soon after treatment onset. Hypermotor seizures disappeared; only sporadic arousals, sometimes with minor motor elements, were observed. Psychometric testing documented improvement in cognitive domains such as visuospatial ability, processing speed, memory, and some areas of executive functions. SIGNIFICANCE: Nicotine appears to be a mechanistic treatment for this specific disorder, probably because of desensitization of the mutated receptors. It may control seizures resistant to conventional drugs for epilepsy and impact socioeducational function in children. This mode of precision therapy should receive more attention and should be available to more patients with uncontrolled CHRNA4-related ADSHE across the age span.

CHRNA2 and Nocturnal Frontal Lobe Epilepsy: Identification and Characterization of a Novel Loss of Function Mutation.

Mutations in genes coding for subunits of the neuronal nicotinic acetylcholine receptor (nAChR) have been involved in familial sleep-related hypermotor epilepsy (also named autosomal dominant nocturnal frontal lobe epilepsy, ADNFLE). Most of these mutations reside in CHRNA4 and CHRNB2 genes, coding for the α4 and ß2 nAChR subunits, respectively. Two mutations with contrasting functional effects were also identified in the CHRNA2 gene coding for the α2 subunit. Here, we report the third mutation in the CHRNA2, found in a patient showing ADNFLE. The patient was examined by scalp EEG, contrast-enhanced brain magnetic resonance imaging (MRI), and nocturnal video-polysomnographic recording. All exons and the exon-intron boundaries of CHRNA2, CHRNA4, CHRNB2, CRH, KCNT1 were amplified and Sanger sequenced. In the proband, we found a c.754T>C (p.Tyr252His) missense mutation located in the N-terminal ligand-binding domain and inherited from the mother. Functional studies were performed by transient co-expression of α2 and α2 Tyr252His , with either ß2 or ß4, in human embryonic kidney (HEK293) cells. Equimolar amounts of subunits expression were obtained by using F2A-based multi-cistronic constructs encoding for the genes relative to the nAChR subunits of interest and for the enhanced green fluorescent protein. The mutation reduced the maximal currents by approximately 80% in response to saturating concentrations of nicotine in homo- and heterozygous form, in both the α2ß4 and α2ß2 nAChR subtypes. The effect was accompanied by a strong right-shift of the concentration-response to nicotine. Similar effects were observed using ACh. Negligible effects were produced by α2Tyr252His on the current reversal potential. Moreover, binding of (±)-[3H]Epibatidine revealed an approximately 10-fold decrease of both Kd and Bmax (bound ligand in saturating conditions), in cells expressing α2Tyr252His. The reduced Bmax and whole-cell currents were not caused by a decrease in mutant receptor expression, as minor effects were produced by α2Tyr252His on the level of transcripts and the membrane expression of α2ß4 nAChR. Overall, these results suggest that α2Tyr252His strongly reduced the number of channels bound to the agonist, without significantly altering the overall channel expression. We conclude that mutations in CHRNA2 are more commonly linked to ADNFLE than previously thought, and may cause a loss-of-function phenotype.