**3.2 GEFS+ and SMEI**

194 Novel Aspects on Epilepsy

On the other hand, some of the disorders display locus heterogeneity where mutations in distinct genes result in the same syndrome. This indicates that other factors beside the primary mutation influence the clinical manifestation of the epilepsy, e.g. environmental factors, developmental events, or differences in inheritance of genetic susceptibility alleles. The latter is supported by mouse models where differences between the genetic backgrounds of two mouse strains influence the severity of a disease caused by the same

Unfortunately, discovery of the responsible gene for an epilepsy syndrome have not led to a prompt understanding of the pathogenesis of the disease. Many of the mutated channels have been characterized in expression systems, but only in some cases have this led to a better understanding of the disease. In other cases this have led to more confusion, as some mutations in a particular channel are found to enhance channel function while others appear to cause a loss of function, even though the clinical manifestation are similar. There are also large discrepancies between results depending on the expression system used to characterize the channels. The mutated channel can e.g. show enhanced function when expressed in *Xenopus laevis* oocytes, while the opposite is shown when expressed in mammalian cells (Meadows et al., 2002). To make it even more difficult, it has been demonstrated that depending on the type of neuron in which a mutated channel is expressed, it can have strikingly different effects on the excitability of the cell (Waxman, 2007). While a mutation can make one type of neuron hyperexcitable, the same mutation can make another neuron hypoexcitable. So changes in neuronal fuction are not necessarily predictable solely from the change in the behaviour of the mutated channel itself, but have to be considered in the cell background in which the mutated channel is expressed. Further, depending on whether a mutated channel mainly is expressed in excitatory or inhibitory neurons, it can have completely opposite effects on the excitability status of the neuronal

The ion channel mutations are bound to cause relative subtle changes in neuronal function. Mutations that cause dramatic changes would likely result in a more severe phenotypes or lethality. The mutations apparently allow normal behaviour under most circumstances, but disturb the equilibrium between excitatory and inhibitory neuronal networks, so that small external perturbations such as fever are sufficient to break the homeostasis and induce

Voltage-gated sodium channels play an essential role in the initiation and propagation of action potentials. These channels open as the membrane depolarizes and inactivate within a few milliseconds of opening. As the membrane polarizes again, the inactivation is removed

Sodium channels are large, multimeric complexes composed of an α subunit and one or more auxiliary β subunits. The α subunit has four homologous domains, each consisting of six transmembrane helices. The β subunit has one transmembrane segment and an extracellular domain with an immunoglobulin-like fold and belongs to the Ig superfamily of cell adhesion molecules (CAMs) (Catterall, 2000). The association with β subunits modulate cell surface expression and localization, voltage-dependence and kinetics of activation and inactivation, as well as cell adhesion and association with signalling and cytoskeletal

sodium channel mutation (Bergren et al., 2005).

**3. Mutations in sodium channel subunit genes** 

and a second depolarizing stimulus is able to reopen the channel.

**3.1 Voltage-gated sodium channels** 

network (Yu et al., 2006).

seizures.

Febrile seizures, i. e. seizures induced by elevated body temperature, affect approximately 3% of children under 6 years of age and are by far the most common seizure disorder. Generalized Epilepsy with Febrile Seizures Plus (GEFS+) is an autosomal dominant epileptic syndrome where the febrile seizures may persist beyond 6 years of age and which may be associated with afebrile generalized seizures (Scheffer and Berkovic, 1997). The disease has a penetrance of approximately 60%. In 1998, GEFS+ was linked to mutation in SCN1B, the voltage-gated sodium channel β1 subunit gene (Wallace et al., 1998). GEFS+ can also result from mutations in the sodium channel α subunit genes SCN1A (Escayg et al., 2000) and SCN2A (Sugawara et al., 2001), and from mutations in the GABRG2 gene which encodes the γ2 subunit of the GABAA receptor (Baulac et al., 2001). Heterozygous mutations in SCN1A can also result in Severe Myoclonic Epilepsy of Infancy (SMEI), also known as Dravet syndrome (Claes et al., 2001). This rare form of epilepsy is characterized by generalized tonic, clonic, and tonic-clonic seizures that are initially induced by fever, light, sound, or physical activity and typically begin around 6-9 months of age. Later, SMEI patients also manifest other seizure types including absence, myoclonic, and simple and complex partial seizures. Psychomotor development stagnates around the second year of life and the patients often respond poorly to antiepileptic drugs. The disorder usually occurs in isolated patients as a result of *de novo* mutations (Claes et al., 2003; Ohmori et al., 2002).

#### **3.3 How mutations in sodium channels can cause seizures**

As sodium channels are responsible for the upstroke of the action potential one might expect that epilepsy-causing mutations in sodium channel genes increase the activity of the channel, thereby allowing increased influx of sodium ions and consequently neuronal hyperexcitability. Indeed, biophysical analyses of the mutant channels have shown that several of the mutations are gain-of-function mutations that increase sodium currents, e. g. by impairing inactivation or by causing a hyperpolarizing shift in the voltage-dependence of the channel (Lossin et al., 2002; Spampanato et al., 2003; Spampanato et al., 2004). The first identified GEFS+ mutation, a C121W missense mutation that disrupts a conserved disulphide bridge in the extracellular Ig domain of the β1 subunit, causes subtle changes in modulation of sodium channel function and alter the ability of β1 to mediate protein-protein interactions that are critical for channel localization (Meadows et al., 2002; Wallace et al., 1998). Electrophysiological and biochemical studies on the mutant C121W β1 subunit coexpressed with NaV1.2 or NaV1.3 have shown that the C121W mutation causes a reduction in current rundown during high-frequency channel activation and increases the fraction of sodium channels that are available to open at subthreshold membrane potentials (Meadows et al., 2002). The mutation is therefore thought to enhance sodium channel function, thereby increasing neuronal excitability and predisposing to seizures.

On the other hand, many of the characterized sodium channel mutations are found to cause attenuation of sodium current (Barela et al., 2006; Lossin et al., 2003; Sugawara et al., 2001).

Epileptic Channelopathies and Dysfunctional

specifically susceptible to febrile seizures.

**4. Mutations in GABAA receptor subunit genes** 

by GABAA receptors (Chebib and Johnston, 1999).

(Oakley et al., 2009).

**4.1 GABA receptors** 

**4.2 GEFS+ and ADJME** 

some patients.

Excitability - From Gene Mutations to Novel Treatments 197

only led to a moderate increase in the respiration rate and did not cause respiratory alkalosis and seizures (Schuchmann et al., 2006). Fever and the accompanying elevated pH and enhanced neuronal activity seem therefore to be the drop that makes the barrel overflow and induce the seizures. As several ion channels are sensitive to changes in pH (Jensen et al., 2005; Prole et al., 2003), it will be interesting to see whether some mutations in sodium channel genes render the channels pH-sensitive, which could make the affected individuals

SMEI patients are normal until their first seizure that typically occurs around 6-9 months of age. This age-specificity may to be related to the time-specific expression of sodium channels. NaV1.1 is undetectable during prenatal and early postnatal development, a stage where NaV1.3 is preferentially expressed. NaV1.3 expression declines at the expression of NaV1.1 increases. An animal model of SMEI has shown that loss of inhibition and seizure onset correlates in time with an increase in NaV1.1 levels and decline in NaV1.3 levels

GABA is the major inhibitory neurotransmitter in the central nervous system. There are three types of GABA receptors: GABAA, GABAB, and GABAC. GABAA and GABAC receptors are ionotropic while GABAB receptors are G-protein coupled and often act by activating potassium channels. Most of the cortical inhibitory effects of GABA are mediated

The GABAA receptors are pentameric chloride channels formed by various combinations of different types of α (α1 to α6), β (β1 to β3), γ (γ1 to γ3), δ, ε, π, θ, and ρ (ρ1 to ρ3) subunits, that each have four transmembrane segments, M1 to M4 (Benarroch, 2007). The most prevalent subunit combination consists of α1β2γ2 (McKernan and Whiting, 1996). The subunit composition determines the functional and pharmacological characteristics of the receptors (Meldrum and Rogawski, 2007; Sieghart and Sperk, 2002). Binding of GABA to the receptor triggers opening of the chloride channel, allowing rapid influx of chloride that hyperpolarizes

As mentioned, GEFS+ can also result from mutation in the GABRG2 gene encoding the γ2 subunit of the GABAA receptor (Baulac et al., 2001). Mutations in the α1 subunit gene (GABRA1) have been linked to Autosomal Dominant Juvenile Myoclonic Epilepsy (ADJME) (Cossette et al., 2002), an idiopathic epilepsy that is not associated with febrile seizures. This disorder typically manifests itself between the ages of 12 and 18 with myoclonic seizures occurring early in the morning and with additional tonic-clonic and absence seizures in

It has been shown that mutations in the γ2 subunit of the GABAA receptor cause retention of the receptor in the endoplasmatic reticulum (ER) (Harkin et al., 2002; Kang and Macdonald, 2004). Similarly, the A322D mutation in the α1 subunit causes rapid ER associated degradation of the subunit through the ubiquitin-proteasome system (Gallagher et al., 2007).

the neuron and thereby decreases the probability of generation of an action potential.

**4.3 How mutant GABAA receptor subunits can cause seizures** 

While it seems like the mild phenotype of GEFS+ mostly is associated with missense mutations that alter the biophysical properties of the channels, the more severe SMEI phenotype is usually caused by nonsense or frameshift mutations that prevent production of functional channels (Claes et al., 2003; Claes et al., 2001; Nabbout et al., 2003; Ohmori et al., 2002). But how can loss-of-function mutations in a sodium channel cause epilepsy when reduced sodium current should lead to hypoexcitability rather than hyperexcitability? The answer seems to be related to the expression pattern of the channels. NaV1.1 is predominantly found in inhibitory interneurons and is thought to conduct most of the sodium current in these cells, whereas excitatory pyramidal neurons express only negligible levels of NaV1.1 (Ogiwara et al., 2007). Catterall and co-workers showed that haploinsufficiency of NaV1.1 channels in heterozygous knock-out mice led to a phenotype resembling that of SMEI (Oakley et al., 2009; Yu et al., 2006). In these mice, sodium currents in GABAergic interneurons in the hippocampus were substantially reduced, whilst the effect in pyramidal cells was much less severe. Loss of one SCN1A copy led to a reduction in action potential number, frequency and amplitude in the interneurons (Yu et al., 2006). Similarly, studies in several animal models carrying nonsense or missense mutations in SCN1A show impaired interneuron function (Martin et al., 2010; Mashimo et al., 2010; Ogiwara et al., 2007; Tang et al., 2009). These studies indicate that functional loss of one copy of SCN1A reduces the inhibitory function of GABAergic interneurons and enhances the excitability of downstream synaptic targets, thereby predisposing to epileptic seizures.

But if this is true, how does the predicted changed NaV1.1 function in many of the patients lead to hyperexcitability when the consequence should be increased GABA action? One possibility is that enhanced sodium current in the interneurons causes too much inhibition, and that this leads to synchronization of the downstream synaptic targets, as has been suggested in the pathogenesis of autosomal dominant nocturnal frontal lobe epilepsy (ADNFL) (Klaassen et al., 2006) (discussed later). Another possibility is that the functional consequences of the mutations in vivo are different from that predicted after in vitro characterization of the mutant channels, and that all of the mutations actually cause a reduction of sodium current in inhibitory neurons. This is supported by studies on knockout mice lacking the β1 subunit (Chen et al., 2004). These mice show downregulated NaV1.1 expression, indicating that β1 function might be necessary for normal expression of NaV1.1. As the inhibitory interneurons seem to be most affected by a reduction in NaV1.1, the consequences of the β1 mutations might be reduced sodium current in interneurons rather than, or in addition to, increased NaV1.2 and NaV1.3 function.

As mutations in SCN1A most often are associated with febrile seizures the mutations seem not to be sufficient to cause spontaneous seizure themselves. Why are the seizures triggered by fever? Why are the seizures most prevalent in young children? And what is the reason for the age-specific onset of SMEI? It is known that an increase in body temperature leads to an increase in the rate of respiration, especially in young children (Gadomski et al., 1994). This increased respiration can cause respiratory alkalosis in the immature brain, and alkalosis of brain tissue can lead to enhanced neuronal activity and to epileptoform activity (Lee et al., 1996). Studies on rat pups showed that seizure activity induced by hyperthermia had a well-defined pH threshold and that a rise in brain pH to the threshold level by injection of bicarbonate could provoke seizures (Schuchmann et al., 2006). By suppressing the alkalosis with a moderate elevation of ambient CO2 to 5%, seizures could be abolished within 20 seconds without affecting body temperature. Bicarbonate-induced pH changes and seizures could also be blocked by elevation of ambient CO2. In older rats, hyperthermia only led to a moderate increase in the respiration rate and did not cause respiratory alkalosis and seizures (Schuchmann et al., 2006). Fever and the accompanying elevated pH and enhanced neuronal activity seem therefore to be the drop that makes the barrel overflow and induce the seizures. As several ion channels are sensitive to changes in pH (Jensen et al., 2005; Prole et al., 2003), it will be interesting to see whether some mutations in sodium channel genes render the channels pH-sensitive, which could make the affected individuals specifically susceptible to febrile seizures.

SMEI patients are normal until their first seizure that typically occurs around 6-9 months of age. This age-specificity may to be related to the time-specific expression of sodium channels. NaV1.1 is undetectable during prenatal and early postnatal development, a stage where NaV1.3 is preferentially expressed. NaV1.3 expression declines at the expression of NaV1.1 increases. An animal model of SMEI has shown that loss of inhibition and seizure onset correlates in time with an increase in NaV1.1 levels and decline in NaV1.3 levels (Oakley et al., 2009).
