Christoph Lossin

A catalog of SCN1A variants

Over the past 10 years mutations in voltage-gated sodium channels (Na(v)s) have become closely associated with inheritable forms of epilepsy. One isoform in particular, Na(v)1.1 (gene symbol SCN1A), appears to be a superculprit, registering with more than 330 mutations to date. The associated phenotypes range from benign febrile seizures to extremely serious conditions, such as Dravets syndrome (SMEI). Despite the wealth of information, mutational analyses are cumbersome, owing to inconsistencies among the Na(v)1.1 sequences to which different research groups refer. Splicing variability is the core problem: Na(v)1.1 co-exists in three isoforms, two of them lack 11 or 28 amino acids compared to full-length Na(v).1.1. This review establishes a standardized nomenclature for Na(v)1.1 variants so as to provide a platform from which future mutation analyses can be started without need for up-front data normalization. An online resource--SCN1A infobase--is introduced.

Chapter 2 Myotonia Congenita

Myotonia is a symptom of many different acquired and genetic muscular conditions that impair the relaxation phase of muscular contraction. Myotonia congenita is a specific inherited disorder of muscle membrane hyperexcitability caused by reduced sarcolemmal chloride conductance due to mutations in CLCN1, the gene coding for the main skeletal muscle chloride channel ClC-1. The disorder may be transmitted as either an autosomal-dominant or recessive trait with close to 130 currently known mutations. Although this is a rare disorder, elucidation of the pathophysiology underlying myotonia congenita established the importance of sarcolemmal chloride conductance in the control of muscle excitability and demonstrated the first example of human disease associated with the ClC family of chloride transporting proteins.

Neuroactive steroids for the treatment of status epilepticus

Benzodiazepines are the current first‐line standard‐of‐care treatment for status epilepticus but fail to terminate seizures in about one third of cases. Synaptic GABAA receptors, which mediate phasic inhibition in central circuits, are the molecular target of benzodiazepines. As status epilepticus progresses, these receptors are internalized and become functionally inactivated, conferring benzodiazepine resistance, which is believed to be a major cause of treatment failure. GABAA receptor positive allosteric modulator neuroactive steroids, such as allopregnanolone, also potentiate synaptic GABAA receptors, but in addition they enhance extrasynaptic GABAA receptors that mediate tonic inhibition. Extrasynaptic GABAA receptors are not internalized, and desensitization of these receptors does not occur during continuous seizures in status epilepticus models. Here we review the broad‐spectrum antiseizure activity of allopregnanolone in animal seizure models and the evidence for its activity in models of status epilepticus. We also demonstrate that allopregnanolone inhibits ongoing behavioral and electrographic seizures in a model of status epilepticus, even when there is benzodiazepine resistance. Parenteral allopregnanolone may provide an improved treatment for refractory status epilepticus.

A human Dravet syndrome model from patient induced pluripotent stem cells

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.

SCN8A encephalopathy: Research progress and prospects

On April 21, 2015, the first SCN8A Encephalopathy Research Group convened in Washington, DC, to assess current research into clinical and pathogenic features of the disorder and prepare an agenda for future research collaborations. The group comprised clinical and basic scientists and representatives of patient advocacy groups. SCN8A encephalopathy is a rare disorder caused by de novo missense mutations of the sodium channel gene SCN8A, which encodes the neuronal sodium channel Nav1.6. Since the initial description in 2012, approximately 140 affected individuals have been reported in publications or by SCN8A family groups. As a result, an understanding of the severe impact of SCN8A mutations is beginning to emerge. Defining a genetic epilepsy syndrome goes beyond identification of molecular etiology. Topics discussed at this meeting included (1) comparison between mutations of SCN8A and the SCN1A mutations in Dravet syndrome, (2) biophysical properties of the Nav1.6 channel, (3) electrophysiologic effects of patient mutations on channel properties, (4) cell and animal models of SCN8A encephalopathy, (5) drug screening strategies, (6) the phenotypic spectrum of SCN8A encephalopathy, and (7) efforts to develop a bioregistry. A panel discussion of gaps in bioregistry, biobanking, and clinical outcomes data was followed by a planning session for improved integration of clinical and basic science research. Although SCN8A encephalopathy was identified only recently, there has been rapid progress in functional analysis and phenotypic classification. The focus is now shifting from identification of the underlying molecular cause to the development of strategies for drug screening and prioritized patient care.

Defective fast inactivation recovery of Nav1.4 in congenital myasthenic syndrome

To describe the unique phenotype and genetic findings in a 57‐year‐old female with a rare form of congenital myasthenic syndrome (CMS) associated with longstanding muscle fatigability, and to investigate the underlying pathophysiology.

Novel HCN2 Mutation Contributes to Febrile Seizures by Shifting the Channel's Kinetics in a Temperature- Dependent Manner

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated currents, known as I h, are involved in the control of rhythmic activity in neuronal circuits and in determining neuronal properties including the resting membrane potential. Recent studies have shown that HCN channels play a role in seizure susceptibility and in absence and limbic epilepsy including temporal lobe epilepsy following long febrile seizures (FS). This study focused on the potential contributions of abnormalities in the HCN2 isoform and their role in FS. A novel heterozygous missense mutation in HCN2 exon 1 leading to p.S126L was identified in two unrelated patients with FS. The mutation was inherited from the mother who had suffered from FS in a pedigree. To determine the effect of this substitution we conducted whole-cell patch clamp electrophysiology. We found that mutant channels had elevated sensitivity to temperature. More specifically, they displayed faster kinetics at higher temperature. Kinetic shift by change of temperature sensitivity rather than the shift of voltage dependence led to increased availability of I h in conditions promoting FS. Responses to cyclic AMP did not differ between wildtype and mutant channels. Thus, mutant HCN2 channels cause significant cAMP-independent enhanced availability of I h during high temperatures, which may contribute to hyperthermia-induced neuronal hyperexcitability in some individuals with FS.

Propofol hemisuccinate suppresses cortical spreading depression

Propofol is a rapidly acting water-insoluble non-barbiturate anesthetic agent that is widely used as an intravenous sedative-hypnotic agent. Anecdotal evidence indicates that propofol may be effective at terminating intractable migraine headache. Cortical spreading depression (CSD) is believed to be the neural correlate of migraine aura and may be a trigger for migraine pain. Agents that block the induction or slow the spread of CSD may be of utility in treating migraine. Here we examined the ability of propofol hemisuccinate (PHS), a water-soluble prodrug of propofol, to affect CSD in mice. For comparison, we examinined dizocilpine, an NMDA receptor antagonist, that is well recognized to inhibit CSD. When administered 15min prior to activation of CSD by KCl application to the cortex, intraperitoneal PHS at doses of 120 and 200mg/kg decreased the number of CSD deflections without an effect on CSD amplitude, and at 200mg/kg caused a 77% reduction in CSD velocity. The minimally-effective dose of PHS (120mg/kg) did not cause sedation or motor impairment and while some animals receiving 200mg/kg did demonstrate motor impariment none exhibited loss-of-righting reflex (anesthesia). Dizocilpine produced comparable inhibition of CSD at doses of 0.5 and 2.5mg/kg. We conclude that acute PHS treatment inhibits CSD. Our results indicate that propofol, or its prodrug PHS, are worthy of further investigation as a treatment for migraine.

Compromised function in the Nav1.2 Dravet syndrome mutation R1312T

Ion channels, specifically voltage-gated sodium channels (Na(v)s), are common culprits in inheritable seizure disorders. Some Na(v) isoforms are particularly susceptible, while others are only weakly associated with neuronal hyperexcitability. Representative of the latter group is Na(v)1.2 (gene name SCN2A): despite its abundance in the brain, Na(v)1.2-related epilepsy is rare and only few studies have been conducted as to the pathophysiological basis of Na(v)1.2 in neuronal hyperexcitability. We here present a detailed functional analysis of Na(v)1.2 mutant, R1312T, which was originally found in a child with Dravet syndrome (formerly known as severe myoclonic epilepsy of infancy or SMEI). Whole-cell voltage clamp analysis revealed clearly compromised function: the mutant channels fast- and slow-inactivated at markedly more negative potentials and recovered from fast inactivation more slowly, which resulted in a use-dependent current reduction to less than 50% of wildtype levels. We also noted a small hyperpolarizing shift in the voltage dependence of activation. Our findings expand the spectrum of abnormal Na(v) channel behavior in epilepsy and raise the question as to how loss-of-function in a sodium channel predominantly expressed in excitatory neurons can lead to hyperexcitability.

Altered fast and slow inactivation of the N440K Nav1.4 mutant in a periodic paralysis syndrome

Objective: To electrophysiologically characterize the Nav1.4 mutant N440K found in a Korean family with a syndrome combining symptoms of paramyotonia congenita, hyperkalemic periodic paralysis, and potassium-aggravated myotonia. Methods: We characterized transiently expressed wild-type and mutant Nav1.4 using whole-cell voltage-clamp analysis. Results: N440K produced a significant depolarizing shift in the voltage dependence of fast inactivation and increased persistent current and acceleration in fast inactivation recovery, which gave rise to a 2-fold elevation in the dynamic availability of the mutant channels. In addition, the mutant channels required substantially longer and stronger depolarization to enter the slow-inactivated state. Conclusions: N440K causes a gain of function consistent with skeletal muscle hyperexcitability as observed in individuals with the mutation. How the same mutation results in distinct phenotypes in the 2 kindreds remains to be determined.