Epileptogenic Channelopathies: Experimental ... - Wiley Online Library

Epilepsia, 48(Suppl. 2):51–64, 2007 Blackwell Publishing, Inc. C International League Against Epilepsy

Epileptogenic Channelopathies: Experimental Models of Human Pathologies Giuliano Avanzini, Silvana Franceschetti, and Massimo Mantegazza Department of Neurophysiopathology, Istituto Neurologico “C. Besta,” Milan, Italy

Summary: The discovery of genetically determined epileptic syndromes associated with specific mutations of genes codifying for subunits of voltage or ligand-activated ion channels highlights the role of ion channels in epileptogenesis. In vitro and in vivo models of channel pathology have been used to define the functional consequence of the mutations identified in human epilepsies. The evaluation of gene-channel mutations based on molecular and physiological techniques have provided significant knowledge on the cellular mechanisms leading to inherited human epilepsies, and possibly to nongenetic human epilepsies

due to “acquired” channel pathologies. We review some of the studies that have explored human epileptic disorders through experimental manipulations of these channels, highlighting some of the difficulties that have arisen using “in vitro” preparations or rodent models. These findings underscore the need for further studies to address the mechanisms involved in mutatedchannel dysfunctions. Key Words: Epilepsy—Seizures—Ion channels—Neurotransmitters—Receptors—Animal models— Heterologous expression—Electrophysiology—Neuron— Transfection.

The role of ion channels in epileptogenesis has been recognized since the 1960s, when it was shown that neurons within experimentally induced epileptic foci have alterations of excitable properties that depend on transmembrane ion currents (Matsumoto and Ajmone-Marsan, 1964). However, it is only recently that we have direct evidence of the relevance of ion channel dysfunctions to naturally occurring epilepsies. An important step in this direction was made possible by a series of experiments on the Drosophila Shaker mutant carried out by the Jans and their colleagues. Their results indicated that a mutation of a gene coding for a potassium channel caused the nerve hyperexcitablility that is responsible for the abnormal behavior of the Shaker mutant (Tempel et al., 1988). A mutation of the SCN4A gene discovered in patients with hyperkalemic periodic paralysis was the first demonstration of a causative relationship between a genetic dysfunction of voltage-sensitive channels and a specific human disorder (Ptàcek et al., 1991). A number of more recent publications have demonstrated that other conditions within the same group of muscle diseases (myotonia, periodic paralyses) are due to mutations of genes coding subunits of voltage-activated ion channels. Moreover, subsequent studies led to the identification of additional mutations of genes coding both voltage- and ligand-activated

ion channels that are involved in a number of diseases, including episodic ataxias, hemiplegic migraine, cardiac arrhythmias, and hyperekplexia (recent reviews include Jurkat-Rott and Lehmann-Horn, 2005; Vicart et al., 2005; Ashcroft, 2006). In recent years, genetically determined channel pathology has been demonstrated or suspected in various forms of idiopathic epilepsies, which presents with transient neuronal hyperexcitability unattributable “to any underlying cause other than a possible hereditary predisposition” (Commission on Classification and Terminology of the International League Against Epilepsy, 1989). ION CHANNELS AND THE PATHOPHYSIOLOGY OF MEMBRANE EXCITABILITY The excitability of neuronal cells depends on the movement of ions through specific cell membrane channels. The biophysical properties of ion channels have been extensively investigated by means of voltage clamp recording techniques, whereas the effect of ion currents on cell membrane potential have been studied by means of single unit current clamp recordings. Studies of various epilepsy models have shown that all of the neurons belonging to an epileptic aggregate consistently discharge in the form of protracted action potential “bursts” lying on a depolarized plateau. This “paradigm” of an elementary epileptic discharge was initially observed in penicillin cortical foci, and was termed paroxysmal depolarization shift (PDS) by Matsumoto and Ajmone Marsan (1964).

Address correspondence and reprint requests to G. Avanzini, Department of Neurophysiopathology, Istituto Neurologico “C. Besta,” Milan, Italy. E-mail: [email protected] doi: 10.1111/j.1528-1167.2007.01067.x

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Similar discharges may be due to intrinsic dysfunctions of various neuronal circuits. In the laboratory, they can be obtained by a number of experimental manipulations that enhance depolarizing ion currents (mainly carried by Na+ and Ca2+ ions) or reduce hyperpolarizing ion currents (mainly carried by Cl− and K+ ions). Thus, a reasonable hypothesis was that epileptogenic mutations of ion channels that cause epilepsy would be identified, and that mutations of these channels would modify their function in a similar fashion as is seen with experimental manipulations. GENETICALLY DETERMINED ION CHANNEL DYSFUNCTIONS AND HUMAN EPILEPSIES We will briefly describe the epileptic syndromes associated with ion channel mutations (for recent reviews, see Avanzini and Franceschetti, 2003; Guerrini et al., 2003; Avanzini et al., 2004; Hirose et al., 2005; Lerche et al., 2005). We will also report some significant evidences obtained expressing mutant channels in heterologous systems (Table 1), studying animal models, and highlighting pros and cons of the experimental models used to evaluate the effects of ion channel mutations. Ion channels are heterooligomeric membrane proteins specifically adapted to regulate transmembrane ion fluxes that are activated by changes in membrane voltage and/or the binding of specific ligands. They typically consist of pore-forming principal subunits and various accessory subunits, and undergo a large number of modulatory and environmental influences. Differential subunit composition and modulation lead to the formation of channels with different relative ion permeabilities, gating properties (opening and closing kinetics, voltage sensitivities, etc.), and ligand affinities. Identification of the molecular structure of the various subunits and of the corresponding genes has revealed a surprising variety of distinct subunits, whose cell typespecific assembly can lead to a considerable number of channel subtypes with different properties (Green and Millar, 1995). The final functional effect of an ion channel mutation therefore depends on the role played by the mutated subunit in specific neuron subtypes, and the role that these neurons play in specific networks. Due to this complex scenario, the results of the functional analysis of epileptogenic channel mutations using in vitro and in vivo models need to be carefully interpreted in order to explain the clinical phenotypes, which raise many questions that are still unanswered. Focal “idiopathic” epilepsies: autosomal dominant nocturnal frontal lobe epilepsy This was an early recognized form of human epilepsy associated with channel mutations (Phillips et al., 1995; Steinlein et al., 1995) that presents with clusters of often stereotyped and brief nocturnal “dystonic” motor seizures Epilepsia, Vol. 48, Suppl. 2, 2007

(Phillips et al. 1995; Steinlein et al., 1995). In different families, ADNFE is associated with different mutations of CHRNA4 (20q13) and CHRNB2 (1q21) genes, encoding the α4 and β2 subunits, respectively, of the nicotinic acetylcholine receptors (nAChR) (see Combi et al., 2004 for a review). The use of mutated channel subunits expressed in cultured cell lines as a means of evaluating the functional consequences of the genetically determined receptor changes has given rise to complex and dyshomogenous data (Bertrand et al., 2002). Data arising from these functional studies suggested that CHRNA4 mutations caused a loss of function of nAChR (i.e., accelerated receptor desensitization leading to a reduced effect of the neurotransmitter on the mutated receptor). Conversely, CHRNB2 mutations have been implicated in an increased sensitivity of the receptor to acetylcholine (i.e., retardation of receptor desensitization or increased affinity to acetylcholine). Moreover, pharmacological and biophysical evaluations of the α4/β2 receptor have shown that different mutations associated with ADNFLE give rise to varying effects, and Rodrigues-Pinguet et al. (2005) have recently proposed an interpretation of channel dysfunction based on evidence of reduced Ca2+ -dependent potentiation of the acetylcholine response due to an alteration in the allosteric activation of the α4/β2 receptor. Thus, the interpretation of the final epileptogenic effect of nAChR channel mutations is still complex; it is however important to keep in mind that cortical nAChR are influenced by different subcortical cholinergic inputs, and dysfunctional channel may sustain an epileptogenic effect by complex mechanisms, such as the fine tuning of neuronal oscillations generating the EEG rhythms and underling cortical synchronization. The rather late occurrence of seizures in ADNFLE, as well as its clinical and EEG characteristics (and dominant transmission), make it substantially different from the more common benign idiopathic focal childhood epilepsies. Despite the advances in our understanding of the functional defects associated with ADNFLE, similar advances has not occurred in other well-known focal epileptic disorders, the prototype of which is benign partial epilepsy with rolandic spikes (Commission on Classification and Terminology of the International League Against Epilepsy, 1989). Benign familial neonatal convulsions Benign familial neonatal convulsions (BFNC) refer to a form of hereditary idiopathic epilepsy in newborns with a dominant autosomal transmission. Seizures typically occur during the first days of life, subsequently becoming less frequent until they spontaneously disappear between the second and 15th week. The neurological picture is normal, as is the subsequent development of brain function. EEG traces are normal or show a picture of “theta pointu

EXPERIMENTAL MODELS OF EPILEPTOGENIC CHANNELOPATHIES alternant.” In different families, BFNC have been associated with mutations of the K+ channel genes KCNQ2 (20q13) or KCNQ3 (8q24). Both K+ channel subunits contribute to the native M-current (Wang et al., 1998), which is a repolarizing current important in controlling hyperexcitability phenomena principally by counteracting subthreshold membrane depolarizations and limiting repetitive firing. The cooperative function of KCNQ2 or KCNQ3 channels in generating M-current explain the association of an identical phenotype with mutations affecting different genes coding different K+ channel subunits (Biervert et al., 1998; Castaldo et al., 2002). Seizures associated with myokymia This rare form of epilepsy is a familial disorder presenting with myokymia-related focal seizures. It is associated with mutations in the KCNA1 gene (12p13) coding for Kv 1.1 channel subunit. The heterologous expression of the mutated channels results in an increased neuronal excitability, due to the impairment of delayed-rectifier type K+ currents (Eunson et al., 2000). Generalized epilepsy and paroxysmal dyskinesia Large conductance Ca2+ -activated K+ channels (BK) are the targets of a recently identified mutation found in individuals affected by generalized epilepsy and paroxysmal dyskinesia, which enhances BK channel functions due to an increase in Ca2+ sensitivity (Du et al., 2005). The authors hypothesized that an enhancement of this K+ current may allow neurons to fire at a faster rate by inducing rapid repolarization of action potentials. Benign familial neonatal-infantile convulsions This autosomal dominant disorder is characterized by clusters of afebrile secondarily generalized partial seizures during the first year of life. It is distinguished from BFNC by its later onset, typically after a few days to a few months of life. Heron et al. (2002) and Berkovic et al. (2004) have found missense mutations in the Na+ channel Nav 1.2 α subunit (gene SCN2A, 2q23-24) in eight BFNIC families, the most common (R1319Q) being found in three families. Functional studies done in transfected neurons indicate that Nav 1.2 mutations modify the gating properties of the channel leading by various mechanisms to a net amplification of Na+ current and thus to an increased depolarizing effect of incoming inputs on neuronal membrane (Scalmani et al., 2006). Generalized epilepsy with febrile seizures Generalized epilepsy with febrile seizures (GEFS+) is a clinical entity first identified by Scheffer and Berkovic (1997), which is characterized by febrile seizures that may or not cease with age (hence the “plus” included in its designation), and are associated with polymorphous afebrile seizures. Wallace et al. (1998) found a mutation involving the SCN1B gene (19q13.1) coding for the β1 subunit of voltage sensitive Na+ channels in a family with a GEFS+

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phenotype. The functional consequences of the mutations were tested in Xenopus oocytes by coexpressing human β1 with the rat Nav 1.2 α subunit. The wild type hβ1 accelerated current decay, whereas the hβ1 mutant was unable to affect it, and thus increased the duration of Na+ current flow consistently with enhanced cell excitability. However, it is not clear yet which is the effect in neurons (see, in vitro models). Shortly after these earliest observations, mutations were identified within the Na+ channel Nav 1.1 α subunit gene (SCN1A, 2q24) that cause a similar human phenotype (Baulac et al., 1999; Escayg et al., 2000b), which probably accounts for most of the GEFS+ families described so far (see, Meisler and Kearney, 2005 for a review). Heterologous expression studies of different mutated Nav 1.1 subunits show a large spectrum of functional effects. Some mutations cause an enhanced fraction (2– 5%) of noninactivating Na+ current (Lossin et al., 2002), that usually accounts for about 1% of the maximal Na+ current peak amplitude, thus becoming capable of reducing the depolarization threshold required for neuronal firing consistently with neuron hyperexcitability. The effects of other mutations are different, but still consistent with hyperexcitability (Spampanato et al., 2003). However, the effects of some mutations are still debated (Alekov et al., 2000; Spampanato et al., 2001; Lossin et al., 2002), and a complete loss of function (see below in SMEI) has been reported for some mutations (Lossin et al., 2003). Severe myoclonic epilepsy of infancy The discovery of SCN1A mutations in patients with the nonfamilial severe myoclonic epilepsy of infancy (SMEI or Dravet syndrome) (Claes et al., 2001) further emphasized the importance of channel pathologies in human epilepsies and suggested that channel mutations may cause neurological disease even in the absence of a positive family history. The SMEI phenotype has a typical natural history: an onset in infancy with long-lasting tonic, clonic, and tonic–clonic seizures, associated or not with fever, followed later in life by myoclonic, tonic–clonic, absence, and simple and complex partial seizures. Since the earliest descriptions, more than 150 mutations have been identified, mainly occurring de novo in the affected children, and many of them are nonsense or frameshift mutations leading to protein truncation, suggesting reduced cell excitability mainly of inhibitory neurons (Wallace et al., 2003; Kanai et al., 2004, see also Meisler and Kearney, 2005). Most SMEI patients are heterozygous, thus the pathogenic mechanism may be a haploinsufficiency. However, functional evaluation in cell lines showed that some SMEI missense mutations may cause a loss of function whereas others a gain of function (Rhodes et al., 2004), thus suggesting that the same epilepsy can be induced by both an increase and a decrease in Na+ channel activity. Another question that needs to be further explored arises from the presence of SCN1A Epilepsia, Vol. 48, Suppl. 2, 2007

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mutations in some apparently healthy parents of a subset of SMEI infants (Nabbout et al., 2003), although parental mosaicisms can explain some of these cases (Gennaro et al., 2006). Furthermore, SCN1A mutations leading to channel loss of function have also been found to be associated with the benign phenotype of GEFS+ (Lossin et al., 2003). All of these observations underlie the complex and incompletely understood relationships between genetically determined dysfunctions and epileptic phenotypes. Simple febrile seizures It has recently been shown that febrile seizures can be caused by a M145T mutation of SCN1A, which cosegregates with the disease in a large family affected by simple febrile seizures inherited as an autosomal dominant trait with high penetrance (>90%). The mutation causes a 60% reduction in current density and a 10 mV positive shift of the activation curve, thus causing channel partial loss of function (Mantegazza et al., 2005b), an effect that is similar to those of some GEFS+ and SMEI mutations. Generalized idiopathic epilepsies with absence seizures Mutations of Ca2+ channel subunits have been linked to various familial epilepsy phenotypes. Familial absence epilepsies can associate with mutations impairing the Cav 2.1 P/Q type alpha subunit (CACNA1A, 19p13) (Imbrici et al., 2004), and sporadic cases of childhood absence epilepsy can associate with mutations of the CaV 3.2 T-type α subunit (CACNA1H, 16p13) (Chen et al., 2003; Vitko et al., 2005). Moreover, mutations in the Ca2+ channel β4 subunit gene CACNB4 (2q22-23) have been identified in patients with idiopathic generalized epilepsy and episodic ataxia which, according to functional studies performed in Xenopus laevis oocytes, cause a small decrease in the inactivation fast time constant of the cotransfected α1 subunit (Escayg et al., 2000a). Gene mutations affecting GABA receptors or Cl− channels Rare GEFS+ phenotypes can associate with mutations affecting the γ 2 subunits of the GABA receptor (GABRG2, 5q31-33) (Baulac et al., 2001). Various other familial epilepsy phenotypes are linked with different mutations of GABA receptor subunits: γ 2 subunit mutations are associated with absence seizures with or without febrile seizures (Wallace et al., 2001; Marini et al., 2003), and α1 subunit (GABRA1, 5q34-35) mutations are associated with idiopathic myoclonic epilepsy (Cossette et al., 2002). The mutations lead to various defects in channel function that are generally consistent with a decreased effect of exogenous GABA, including a decrease in the amplitude of GABA-induced currents (Baulac et al., 2001), altered channel gating (Fisher, 2004), and probably reEpilepsia, Vol. 48, Suppl. 2, 2007

duced surface expression of functional receptors (Kang and Macdonald, 2004; Hales et al., 2005). Mutations affecting the gene CLCN2 (3q27-28) coding a voltage gated Cl− channel possibly involved in physiological GABA-dependent inhibition have also been reported in families with various types of idiopathic generalised epilepsies with absence and convulsive seizures (Haug et al., 2003). The mutated channels show complete loss of function or altered voltage-dependent gating possibly causing impaired Cl− homeostasis or membrane depolarization, and favoring hyperexcitability. EXPERIMENTAL MODELS OF EPILEPTOGENIC CHANNELOPATHIES In vitro models As briefly reviewed above, a considerable amount of data on the functional effects of epileptogenic mutations is now available. As it is difficult to manipulate genes efficiently in intact cells, channels are often expressed in “heterologous” (nonneuronal) cells that have few or no endogenous currents in order to study the expressed channels in isolation. These systems are relatively easy to use and a large number of mutants can be screened. The results obtained with these experimental models indicate that some epileptogenic human mutations lead to loss of function of channels that are important for membrane repolarization or for counteracting membrane depolarizations, whereas mutations causing a gain of function can be found in channels that generate depolarizing currents, in line with the expected induction of hyperexcitability phenomena. However, several mutations have opposite effects that are consistent with neuronal hypoexcitability. Moreover, some mutations that cause distinct epilepsies have similar functional effects. Some of these controversial results may depend on the experimental model. In the following paragraphs, we will try to highlight how the expression system can influence the results of studies aimed at revealing the functional effects of epileptogenic mutations. Xenopus oocytes The oocytes of the African clawed frog Xenopus laevis have been widely used to express heterologous proteins ever since Gurdon et al. (1971) demonstrated that mRNA injected into the cytoplasm can be translated into functional proteins (Fig. 1). About 10 years later, it was shown that a variety of nervous system receptors and channels could also be functionally expressed in oocytes (Barnard et al., 1982; Gundersen et al., 1983). Oocytes are still used as an expression system in many laboratories because they have a number of distinct advantages. They can be easily removed without the need for animal sacrifice, furthermore, they are large (a diameter of approximately 1–1.2 mm), quite resistant, and can be kept alive in vitro for a few weeks using relatively simple and

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FIG. 1. Schematic diagram of the experimental models used in the study of epileptogenic channelopathies.

inexpensive facilities. They have only a few endogenous currents, are easily impaled with microelectrodes, allow long and stable recordings, and it is possible to control the ratio of different expressed subunits injecting fixed amounts of RNA. The traditional two-electrode voltageclamp technique, which is used because oocytes are too large for whole-cell patch-clamp experiments (Sakmann and Neher, 1995), does not allow fast clamping of the membrane potential, thus limiting the evaluation of fast voltage-dependent properties of the channels. However, this problem can be overcome by making patch-clamp recordings of macroscopic currents from a relatively large patch of oocyte membrane (macropatch), which allows a good signal-to-noise ratio and fast voltage clamping (Sakmann and Neher, 1995). Moreover, the cut-open oocyte voltage-clamp technique (Stefani and Bezanilla, 1998) allows both fast voltage clamping and control of the composition of the intracellular medium. The most serious disadvantage of the oocyte system is its cell background because the expressed channels may interact with endogenous proteins and undergo posttranslational modifications that may be different from those occurring in mammalian cells It is therefore not surprising that the ion channels expressed in Xenopus oocytes often show different properties from those studied in mammalian cell lines (see, e.g., Meadows et al., 2002). Mammalian cell lines Heterologous expression in mammalian cell lines transfected with cDNA makes it possible to study the proper-

ties of the channel of interest while preserving a mammalian cell background (Fig. 1). Two cell lines have become standards for ion channel studies: Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK) cells (Thomas and Smart, 2005; Gamper et al., 2005). They both have epithelial-like morphology and very small endogenous currents, and are easy to maintain and transfect using inexpensive methods. They are relatively easy to use in electrophysiological experiments (typically wholecell patch-clamp recordings), thus allowing a relatively rapid screening of mutants, fast and accurate clamping of membrane voltage, and good control of the composition of the intracellular medium. In particular, HEK cells have been used because they have a massive expression of exogenous proteins (particularly in the case of the derived tsA-201 cell line, which expresses simian virus 40 large tumor antigen) and a human cell background (Lossin et al., 2002). However, striking differences in channel properties can be seen between different mammalian cell lines or even different subclones. In an attempt to give significant examples of the problems encountered when studying the properties of mutated channel subunits, we will focus on Na+ channels, which are major targets of epileptogenic mutations and very sensitive to the different cell backgrounds of mammalian cell lines (Baroudi et al., 2000; Chen et al., 2000; Cummins et al., 2001; Mantegazza et al., 2005a). Many epileptogenic Na+ channel mutations have unclear and cell line-dependent effects: for example, the GEFS + R1648H mutation of the Nav 1.1 Na+ channel Epilepsia, Vol. 48, Suppl. 2, 2007

Loss of function Gain of function

Delayed rectifier IKCa (BK)

KCNA1

KCNMA1

Epilepsia, Vol. 48, Suppl. 2, 2007 ICa (P/Q) ICa (T)

SCN1A

SCN1A

SCN1A

SCN1A

CACNA1A

CACNA1H

CACNB4

GEFS+ type 2

SMEI

ICEGTC

FS

Absence epilepsy and episodic ataxia CAE

IGE and episodic ataxia

ICa

INa

INa

INa

INa

INa

SCN1B

GEFS+ type 1

INa

SCN2A

BFNIC

Gain of function

Gain or loss of function

Loss of function

Loss of function




Gain of function

Gain of function

Loss of function

Focal familial seizures and myokymia Generalized epilepsy & paroxysmal dyskinesia

M-current

KCNQ2 KCNQ3

Effect on the current

BFNC

Affected current

Affected gene

Human epilepsy Decreased expression or modifications of gating kinetics that reduce K+ M-current induced hyperpolarization Decreased delayed rectifier K+ current by various mechanisms Enhanced Ca2+ sensitivity (cell hyperexcitability may be due to rapid action potential repolarization and enhanced recurrent firing) Increase of current by various modifications of voltage dependence of gating Variable according to the expression system, often loss of modulation of INaT inactivation Variable according to the mutation, expression system and cDNA used Slowed time course of INaT inactivation and faster recovery from inactivation Decreased use dependent inactivation Enhanced INaP fraction and decreased fast inactivation of INaT Hyperpolarizing shift of both INaT activation and inactivation causing a hyperpolarizing shift of window current Depolarizing shift of INaT activation Reduced current and enhanced recovery from slow INaT inactivation Complete loss of function (no current) Depolarizing shift of INaT steady-state inactivation due to altered interaction with beta1 subunit No current Enhanced INaP fraction No current No current Various effects on gating properties according to the mutation Decreased current, positive shift voltage dependence of activation Decreased P/Q Ca2+ current by reduced membrane targeting Various effects on gating properties of T type Ca2+ channels Decrease in the fast inactivation time-constant when coexpressed with alpha subunit

Main functional mechanism

TABLE 1. Examples of functional effects of epileptogenic mutations of ion channels

Escayg et al., 2000a

Vitko et al., 2005

Imbrici et al., 2004

Mantegazza et al., 2005b

Rhodes et al., 2005

Lossin et al., 2003 Rhodes et al., 2004

Spampanato et al., 2004

Lossin et al., 2003

Spampanato et al., 2003

Spampanato et al., 2001 Lossin et al., 2002

Alekov et al., 2000

continued.

Wallace et al., 1998; Meadows et al., 2002

Scalmani et al., 2006

Du et al., 2005

Eunson et al., 2000

Biervert et al., 1998; Castaldo et al., 2002

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BFNC, benign familial neonatal convulsions; BFNIC, benign familial neonatal-infantile convulsions; GEFS+, generalized epilepsy with febrile seizure plus; SMEI, severe myoclonic epilepsy in infancy; ICEGTC, intractable childhood epilepsy with generalized tonic–clonic seizures (a disorder similar to SMEI); FS, febrile seizures; IGE, idiopathic generalized epilepsy; CAE, childhood absence epilepsy; JME, juvenile myoclonic epilepsy; ADNFE, autosomal dominant nocturnal frontal lobe epilepsy; INaT, transient sodium current; INaP, persistent sodium current; nAChR, nicotinic acetylcholine receptor.

Gain of function nAChR ADNFLE type 3

CHRNB2

Loss of function nAChR CHRNA4 ADNFLE type 1

IGABA IGABA CAE & FS JME

GABRG2 GABRA1

Loss of function Loss of function

Slower desensitization Increase in acetylcholine sensitivity

Bertrand et al., 2002; Combi et al., 2004 DeFusco et al., 2000 Phillips et al., 2001

Baulac et al., 2001; Kang and Macdonald, 2004; Hales et al., 2005 Wallace et al., 2001 Cossette et al., 2002; Fisher, 2004 IGABA GEFS+ type 3

GABRG2

Loss of function

Complete loss of function causing decreased transmembrane Cl− gradient (and GABA inhibition) Changes in voltage-dependent gating (membrane depolarization?) Decreased current amplitude by reduced membrane targeting and receptor assembly Loss of benzodiazepine sensitivity Reduced GABA sensitivity and altered channel gating Various effects Loss of function ICl CLCN2 IGE with absences and convulsions

Effect on the current Affected current Affected gene Human epilepsy

TABLE 1. Continued

Main functional mechanism

Haug et al., 2003

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α subunit has been examined in three expression systems with different results (Alekov et al., 2000; Spampanato et al., 2001; Lossin et al., 2002). Furthermore, the R1648C substitution at the same position causes SMEI, but has similar effects to those of R1648H when expressed in HEK cells (Rhodes et al., 2004). The effects of the Na+ channel β1 subunit are also highly variable and strictly depend on the expression system used, thus complicating the identification of its functions (Isom et al., 1994; Qu et al., 2001; Meadows et al., 2002; Tammaro et al., 2002). The GEFS+ C121W mutant of the β1 subunit is a loss of function mutation because, in all of the systems in which it has been studied, it cannot modulate principal α subunits (Meisler and Kearney, 2005); however, its effects on cell excitability are not yet fully understood because those of the wild type β1 are still unclear. These differential properties and controversial results may be due to the fact that these cell lines express a variety of neuronal genes, including many accessory subunits of ion channels that could differently modulate exogenous channels and thus produce cell linespecific properties (Thomas and Smart, 2005; Gamper et al., 2005). Moreover, different cell lines can also have specific membrane lipid compositions, which may be surprisingly important in setting the functional properties of ion channels (Oliver et al., 2004). One intriguing possibility is that this variety of effects may not just be an experimental artifact, but reproduces typical pathophysiological neuronal variability. Each neuronal subtype has its own specific expression pattern and lipid environment, and this could give to the expressed channels neuronal subtype-specific properties, and it is interesting to note that recordings from mice not expressing functional Nav 1.6 Na+ channels have indeed revealed neuronal subtype-specific effects (Raman and Bean, 1997; Maurice et al., 2001). Moreover, mutations may modify the subcellular distribution of ion channels, which is an important parameter in setting the input–output relationship of a neuron (Lai and Jan, 2006), but this effect may be undetectable in cell lines. Ion channel functional properties may also vary according to the neuronal subcellular compartment, due to specific interactions and modulations that could take place, for example, in specialized plasmamembrane microdomains (Galbiati et al., 2001; Martens et al., 2004). Furthermore, cell lines do not allow the direct study of the effect that the mutations have on synaptic transmission. Thus, cell lines do not completely reproduce neuronal genetic background, cell variety, and subcellular specialization. Primary cultured neurons Transfected neurons in primary cultures provide an expression system that makes it possible to study exogenous channels while preserving neuronal properties, background, and variety (Fig. 1). Neurons isolated from various Epilepsia, Vol. 48, Suppl. 2, 2007

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G. AVANZINI ET AL. TABLE 2. Examples of in vivo models of genetically determined channel dysfunctions

Channel Ca2+ K+ K+ K+ Na+ Na+ Na+

Gene defect

Affected current

Human epilepsy

Mouse epilepsy

CaV 2.1 (CACNA1A) knockout KCNQ2 knockout KCNQ2 dominant-negative

P/Q type

Absence seizures

Absence seizures

Song et al., 2004

M-type M-type

BFNC BFNC

Watanabe et al., 2000 Peters et al., 2005

Kv 1.1 (KCNA1) knockout Nav 1.1 (SCN1A) deletion Nav1.2 (SCN2A) mutation β1 (SCN1B) knockout

Delayed rectifier

Seizures and myokymias

Increased sensitivity to convulsants Seizures (behavioral disturbances & changes in hippocampus morphology) Increased susceptibility to seizures

Rho et al., 1999

Na+ current

SMEI

Spontaneous seizures

Yu et al., 2006

Persistent Na+ current Na+ current

GEFS+

Spontaneous seizures and hippocampal neuronal loss Spontaneous seizures (ultrastructural changes in glioneuronal interactions)

Kearney et al., 2001

GEFS+

brain areas can now be efficiently transfected and, in neocortical and hippocampal cultures, it is also easy to identify and make selective recordings of excitatory neurons (with typical pyramidal shapes) or inhibitory interneurons (bipolar, and positive for glutamic acid decarboxylase) (Scalmani et al., 2006). This should therefore be the system of choice for reproducing differential neuronal cell backgrounds, studying the biophysical properties of wild type and mutant subunits, and investigating the effects of the mutations on synaptic transmission, but it does of course have some disadvantages. First of all, it may be difficult to study exogenous transfected channels in isolation because neurons express a plethora of endogenous channels, but this is not an insurmountable problem because exogenous channels can be selectively recorded by transfecting engineered channels that are resistant to specific blockers (Cummins et al., 2001). Secondly, cultured central nervous system neurons develop long processes that make good space-clamping of membrane voltage difficult; however, as in the case of Xenopus oocytes, this can be overcome by means of macropatch recordings (Scalmani et al., 2006). However, the real disadvantage of this system is that electrophysiological experiments are more difficult than in the case of cell lines, which makes rapid mutant screening something of a problem. Moreover, as in the other systems, exogenous channels are usually highly overexpressed at nonphysiological levels. In vivo models In vivo models of genetically determined epilepsies should better reproduce real physiopathological conditions and complexity, and are often considered the best experimental model although they do not allow an efficient screening of mutants (Fig. 1, Table 2). Animal models due to primary membrane channel dysfunction consist of gene-targeted mice generated by means of genetic manipulations, and rodent strains “spontaneously” presenting with seizures in which genetic evaluations have Epilepsia, Vol. 48, Suppl. 2, 2007

Chen et al., 2004

discovered specific ion channel dysfunctions. There are currently relatively few gene-targeted models replicating human mutations probably because they are costly and time-consuming to prepare, and sometimes fail to reproduce human diseases. The characteristics and the methods of selection/generation of spontaneous and gene-targeted murine models of genetically determined epilepsies have been extensively reviewed by Burgess (Burgess, 2006) and by Noebels (Noebels, 2006), pointing out the limits and the value of these animal models in effectively replicate human epileptic phenotypes. Several possible regulatory steps may in fact influence the expression and the properties of the mutated channel differently than in humans. Moreover, other channels may vicariate the dysfunctional ones, in particular just in a definite and species-specific developmental time window. This is especially important because many genetic epilepsies in humans present with an age-related phenotype, which probably result from the complex developmental phenomena controlling membrane excitability in different species. In general, channel modulation, interactions, and functions are certainly influenced by the genetic background that, despite many similarities, is obviously different between mice and humans, and it is often uneven also in different mice strains. In spite of these limitations, genetargeted in vivo models of human channelopathies should be considered as a fundamental tool for understanding the role of channel dysfunctions also in case of incomplete replication of the human phenotype, because they allow the evaluation of the effect of the mutation both at the cellular and at the network level, preserving the complexity of the nervous system. We will summarize here the phenotype characteristics of some in vivo animal models in order to compare them with the phenotypes of human disorders and evaluate their contribution to the understanding of genetically determined human epilepsies.

EXPERIMENTAL MODELS OF EPILEPTOGENIC CHANNELOPATHIES Ca2+ channel mutations Ca2+ channel mutations account for most of the rodent strains that spontaneously present with seizures inherited as an autosomal recessive trait (Burgess and Noebels, 1999; Fletcher and Frankel, 1999). Most spontaneous seizures show ictal EEG-behavioral patterns similar to those of human epileptic syndromes with simple “absence” seizures. However, the discrepancies include the persistence of absence seizures in adult and old rodents, and the frequent association with other neurological symptoms, whereas human epilepsies with absence seizures often remit spontaneously and occur in neurologically healthy subjects. Nevertheless, their generalized onset and ictal EEG characteristics, high seizure recurrence rates and ictal behavior provide a faithful reproduction of human absence epilepsies. Four spontaneous autosomal recessive mouse mutants (tottering: tg/tg, leaner: tgla /tgla , rolling Nagoya: tgrol /tgrol and rocker: tgrkr /tgrkr ) show loss of function mutations in the CaV 2.1 α 1 subunit that cause reduced P/Q current density in neurons and, in principle, imply reduced cell excitability as P/Q-type Ca2+ channels play a dominant role in synaptic transmission (see, review by Pietrobon, 2005). However, this observation seems to diverge from previous evidence indicating an enhanced T-type Ca2+ current (flowing through CaV 3.1 channels) in the thalamocortical neurons of various rodent models of absence seizures (Zhang et al., 2002). Indeed, T-currents can sustain rhythmic burst firing in thalamocortical relay neurons and represent an important cell substrate of the synchronization occurring during absence seizures (Avanzini et al., 1999, 2000). One possible solution to this dilemma is the finding of reduced excitatory neurotransmission (coupled with unimpaired transmission at inhibitory synapses) to thalamocortical neurons in tg/tg mice, which is due to CaV 2.1 channel loss of function. The prominent inhibitory neurotransmission may thus give rise to the excessive hyperpolarization of thalamocortical neurons, which may allow the Tcurrent to activate and promote burst firing and thalamic synchrony. In line with these findings and interpretations, the thalamocortical neurons of mice lacking CaV 3.1 channels do not show burst firing (Kim et al., 2001). Furthermore, double knockout CaV 2.1/CaV 3.1 and double mutant tg/tg /CaV 3.1−/− mice do not show spike and wave discharges, thus providing in vivo evidence that CaV 3.1 channels play a crucial role in the genesis of spontaneous absence seizures (Zhang et al., 2002). However, the recent finding of typical spike and wave discharges in CaV 3.1 heterozygotes of CaV 2.1−/− background (CaV 2.1−/− /CaV 3.1+/− ) mice showing lower amplitude T-type currents in thalamocortical neurons than those observed in wild-type mice has suggested that an enhanced T-type current is not necessary for the genesis of absence seizures (Song et al., 2004).

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The spontaneously “lethargic” (lh) mutant mouse shows ataxia and lethargy followed by the onset of spontaneous focal motor and absence seizures similar to those of human petit mal epilepsy and those seen in “tottering” mice. This lh phenotype arises from loss of function mutations of β4 (CACNB4) calcium channel subunits (Burgess and Noebels, 1999). Ca2+ channels are involved in many more cell functions than the direct control of membrane excitability due to voltage-activated Ca2+ currents. These include signal transduction, cell differentiation/proliferation and synaptogenesis, and so the role of mutated Ca2+ channels in generating the absence epilepsy phenotype may include more complex phenomena, which would also explain the associated neurological dysfunction (and apparently neurodegenerative aspects) found in mutated animal models but unusual in spontaneous human epilepsies. K + channel mutations K+ channels play a critical role in limiting neuronal excitability, and mutations in specific voltage-gated K+ channels have been associated with hyperexcitable phenotypes in both humans and animals. Mutations in the K+ channel genes for members of the KCNQ channel family (KCNQ2/KCNQ3) are associated with the human epileptic syndrome, BFNC (Biervert et al., 1998; Wang et al. 1998). All these mutations lead to a loss of function, and therefore reduce the efficiency of the hyperpolarizing voltage-dependent currents carried by the K+ ions flowing through the channels. However, the complexity of voltagedependent K+ currents due to the vast number of different channel subunits expressed in the mammalian brain may make the situation more complicated than expected because of the possible surrogate effect of “healthy” channel subunits. On the other hand, replacement of the hyperpolarizing function by other K+ channel subunits or other hyperpolarizing (e.g., GABA-mediated) mechanisms must be assumed to explain the transient epileptic manifestations of infants with KCNQ mutations, which quickly fade away during early postnatal development. The heterotetramer products of KCNQ genes form Mchannels that regulate the threshold of electrical excitability in neurons. M-currents activate at close to resting membrane potential, counteract membrane depolarizations, and are sensitive to muscarinic (M) acetylcholine receptor agonists. Homozygous pups prepared by disrupting the mouse KCNQ2 gene via gene targeting die of pulmonary atelectasis within a few hours of birth, whereas heterozygous mice with decreased KCNQ2 channel expression do not present spontaneous seizures but are significantly hypersensitive to epileptogenic manipulations based on the classical convulsant pentylenetetrazol (Watanabe et al., 2000). Another mouse model, which has been more recently obtained by means of the conditional expression Epilepsia, Vol. 48, Suppl. 2, 2007

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of dominant-negative KCNQ2 subunits in brain, presents with spontaneous seizures and behavioral hyperactivity. As expected, in the case of an absent (or reduced) Mcurrent, the hippocampal CA1 pyramidal neurons obtained from the mutated mice show increased excitability, reduced spike-frequency adaptation, attenuated afterhyperpolarization, and reduced intrinsic subthreshold theta-resonance. Moreover, the restriction of transgene expression to defined developmental periods has revealed that M-channel activity is critical for the development of normal hippocampal morphology, thus suggesting that these types of K+ channels may play a part in physiological brain maturation (Peters et al., 2005). The KCNA1 gene mutations discovered so far sustain various neurological disorders, but rarely an epileptic phenotype (Eunson et al., 2000). Different mutations probably unevenly affect the function of the delayed-rectifier type of K+ current flowing through these channel types, thus leading to different clinical expressions. Knockout mice lacking the voltage-gated Shaker-like K+ channel Kv 1.1 α-subunit (the homologue of human KCNA1) show recurrent spontaneous seizures during postnatal development, and heterozygous Kv 1.1+/− mice show indirect evidence of increased early seizure susceptibility (Rho et al., 1999). Na+ channel mutations Most human epileptogenic channel mutations involve the Nav 1.1 (SCN1A) Na+ channel α subunit (see, review by Meisler and Kearney, 2005). SCN1A mutations may be associated with “benign” phenotypes with seizures limited to early infancy, intermediate phenotypes (GEFS+), or severe phenotypes mainly represented by SMEI, and so a still open question is the relationship between the dysfunctional effects of channel mutations and the severity of the associated human epilepsy. As mentioned above, it has been suggested that many SCN1A mutations associated with SMEI give rise to a loss of function of Nav 1.1. There are few data concerning the in vivo dysfunctions of SCN1A mutations that might help to clarify this point, but encouraging results have been obtained from the evaluation of heterozygous SCN1A knockout mice presenting with severe seizures, probably caused by decreased excitability of GABAergic neurons, and with a very high, postnatal mortality rate (Yu et al., 2006). Only a few epilepsy mutations have been detected in SCN2A in patients with benign familial neonatal-infantile seizures (Sugawara et al., 2001; Heron et al., 2002), and only one truncation mutation of SCN2A has been identified in a patient with intractable epilepsy resembling SMEI (Kamiya et al., 2004). Focal motor seizures occur in a mouse with a SCN2A mutation that significantly increases the persistent fraction of Na+ currents and, in the same mice model, Nissl-stained sections of hippocampus area CA3 reveal extensive neuronal cell loss, thus suggesting Epilepsia, Vol. 48, Suppl. 2, 2007

that neurodegenerative phenomena take place because of the gain of function mutation (Kearney et al., 2001). SCN1B mutations (involving the β1 subunit of Na+ channels) may also be associated with the human GEFS+ phenotype, although they are apparently rarer than mutations involving α subunits. β1 subunit knockout mice appear ataxic and show spontaneous generalized seizures (Chen et al., 2004) that may be caused by a selective modification of the expression levels of other sodium channel subunits in a subset of neurons. Moreover, ultrastructural changes found at the level of the Ranvier nodes in the knockout mice suggest that the β1 subunit plays a role in controlling the maturation of axo-glial communication. Cl− channel mutations Knockout mice lacking ClC-2 Cl− channel, whose mutations have been found in patients affected by idiopathic generalized epilepsies with absence and convulsive seizures (Haug et al., 2003), do not show seizures or any other paroxysmal abnormality, but suffer severe retina and testes degeneration probably due to defective ionic homeostasis with no obvious changes in cell excitability (Bosl et al., 2001). Neurotransmitter-activated ion channels The availability of animal models aimed at reproducing impaired receptor function associated with human epilepsies is extremely poor. Mutations of the α4 or β2 subunits of the neuronal nicotinic acetylcholine receptor (nAChR) is associated with human autosomal dominant frontal lobe nocturnal epilepsy (ADNFE). α4 nAChR knockout mice do not present spontaneous seizures or show any EEG differences in comparison with wild-type mice, however, in response to proconvulsant manipulations; they do show a greater number of generalized clonic seizures and epileptic EEG abnormalities, thus suggesting that naive nAChR receptor subunits protect against convulsants (Wong et al., 2002; McColl et al., 2003). About GABA receptors (whose mutations are associated with GEFS+, absence seizures and idiopathic myoclonic epilepsy phenotypes in humans), various mice models show secondary defective GABA transmission or interneurons, but there are very few data relating to models obtained by means of the targeted manipulation of genes coding for GABA receptors. Knockout mice for the α1 subunit show an increased sensitivity to the GABA-antagonist bicuculline (Kralic et al., 2002), and knockout mice for the β3 subunit of the GABAA receptor show spontaneous epilepsy and hyperactivity (DeLorey and Olsen, 1999), and have been proposed as a model for the severe developmental disorders associated with Angelman’s syndrome, which is known to be of genetic origin. However, no genetically targeted models mimic human idiopathic epilepsies that arise from mutations of GABA receptor subunits.

EXPERIMENTAL MODELS OF EPILEPTOGENIC CHANNELOPATHIES CONCLUSIONS The demonstration that the structural alterations of ligand- and voltage-gated channels found in some human epilepsies can lead to potentially epileptogenic changes in ionic currents is a major step forward our understanding of epileptogenesis. The opportunity to combine molecular and physiological techniques in in vivo and in vitro evaluations of the dysfunctional effects due to gene-channel mutations provides an extraordinary means of directly understanding the exact epileptogenic mechanisms leading to inherited human epilepsies. These types of investigations may also open a window on nongenetic human epilepsies probably due to “acquired” channel pathologies. However, despite these advantages, further studies are necessary in order to acquire additional information on the final effect of channel mutations and prepare therapeutic strategies aimed at specifically correcting/counteracting the hyperexcitability phenomena due to dysfunctional channels. In fact, after the earliest indications of simple links between a genetic defect and its consequences, the scenario has become more complicated than expected. The evident advantage of in vitro preparations is the possibility to directly evaluate in a simple system the modifications of the biophysical properties of the channels caused by the mutations. They also allow a relatively fast screening of mutants. However, major concerns derive from the evidence of a possible modulating effect of the cell background on the properties of wild type and mutant channels. A future evolution of in vitro studies should include control experiments performed expressing the protein of interest in neurons, thus preserving the physiological cell background and allowing the study of the effect of the mutations in different neuronal subtypes (e.g., excitatory vs. inhibitory neurons prepared from different brain structures). Gene-targeting in mice for generating animal models of genetic human epilepsies allows the study of the effect of a given mutation not only in individual neurons but also in neuronal networks and at the behavioral level. The main limit of in vivo models derive from the fact that a specific genetic background (a species-specific pattern of modulations, gene expression, and protein interactions) can give rise to phenotypes that are often difficult to compare with the human ones. In comparison with humans, both spontaneous and gene-targeted murine models of human epilepsies often present with seizure patterns that are either more severe or milder (sometime disclosed only by means of pharmacological manipulation), or with seizures associated with other behavioural or neurological defects that are absent in human epilepsies. In general, it should be kept in mind that models of genetic epilepsies suffer from a degree of limitations that is comparable to that of other models of in vivo epilepsies (Schwartzkroin and Engel, 2006). Thus, their phenotype has to be carefully evaluated

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and the goal of the experiments has to be well identified. Moreover, animal models should not be considered a perfect replication of human disorders. Keeping in mind all the limits deriving from the experimental manipulations, the critical evaluation and integration of the results obtained in different experimental models will probably help to completely disclose the effects of epileptogenic mutations The observation of channel mutations in infants with severe (formerly cryptogenic) epilepsies is especially important and may link channel dysfunction with neurodegenerative phenomena. Furthermore, the finding that some mutations of the channels with excitatory functions associated with severe epilepsies lead to nonfunctional channels indicates the importance of evaluating the results of channel mutations in experimental models bearing in mind the biological background and impact of the channel dysfunction on different neuronal subtypes and different neuronal circuitries. REFERENCES Alekov A, Rahman MM, Mitrovic N, Lehmann-Horn F, Lerche H. (2000) A sodium channel mutation causing epilepsy in man exhibits subtle defects in fast inactivation and activation in vitro. Journal of Physiology 15(529 Pt 3):533–539. Ashcroft FM. (2006) From molecule to malady. Nature 440:440–447. Avanzini G, Franceschetti S. (2003) Cellular biology of epileptogenesis. Lancet Neurology 2:33–42. Avanzini G, de Curtis M, Pape HC, Spreafico R. (1999) Intrinsic properties of reticular thalamic neurons relevant to genetically determined spike-wave generation. In Delgado-Escueta AV, Wilson WA, Olsen RW, Porter RJ (Eds.) Jasper’s Basic Mechanisms of the Epilepsies, Third ed. Lippincott Williams & Wilkins, Philadelphia, pp. 297–309. Avanzini G, Panzica F, de Curtis M. (2000) The role of the thalamus in vigilance and epileptogenic mechanisms. Clinical Neurophysiology 111(suppl 2):S19–26. Avanzini G, Franceschetti S, Avoni P, Liguori R. (2004) Molecular biology of channelopathies: impact on diagnosis and treatment. Expert Review of Neurotherapeutics 4:519–539. Barnard EA, Miledi R, Sumikawa K. (1982) Translation of exogenous messenger RNA coding for nicotinic acetylcholine receptors produces functional receptors in Xenopus oocytes. Proceedings of the Royal Society of London. Series B. Containing Papers of a Biological Character 215:241–246. Baroudi G, Carbonneau E, Pouliot V, Chahine M. (2000) SCN5A mutation (T1620M) causing Brugada syndrome exhibits different phenotypes when expressed in Xenopus oocytes and mammalian cells. FEBS Letters 467:12–16. Baulac S, Gourfinkel-An I, Picard F, Rosenberg-Bourgin M, Prud’homme JF, Baulac M, Brice A, LeGuern E. (1999) A second locus for familial generalized epilepsy with febrile seizures plus maps to chromosome 2q21-q33. American Journal of Human Genetics 65:1078–1085. Baulac S, Huberfeld G, Gourfinkel-An I, Mitropoulou G, Beranger A, Prud’homme JF, Baulac M, Brice A, Bruzzone R, LeGuern E. (2001) First genetic evidence of GABA(A) receptor dysfunction in epilepsy: a mutation in the gamma2-subunit gene. Nature Genetics 28:46–48. Berkovic SF, Heron SE, Giordano L, Marini C, Guerrini R, Kaplan RE, Gambardella A, Steinlein OK, Grinton BE, Dean JT, Bordo L, Hodgson BL, Yamamoto T, Mulley JC, Zara F, Scheffer IE. (2004) Benign familial neonatal-infantile seizures: characterization of a new sodium channelopathy. Annals of Neurology 55:550–557. Bertrand D, Picard F, Le Hellard S, Weiland S, Favre I, Phillips H, Bertrand S, Berkovic SF, Malafosse A, Mulley J. (2002) How mutations in the nAChRs can cause ADNFLE epilepsy. Epilepsia 43(suppl 5):112–122.

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