Current Literature In Basic Science
TALE of an SCN8A-Associated Epileptic Encephalopathy Mouse Model
Convulsive Seizures and SUDEP in a Mouse Model of SCN8A Epileptic Encephalopathy. Wagnon JL, Korn MJ, Parent R, Tarpey TA, Jones JM, Hammer MF, Murphy GG, Parent JM, Meisler MH. [published online ahead of print September 26, 2014]. Human Molecular. pii: ddu470.
De novo mutations of the voltage-gated sodium channel gene SCN8A have recently been recognized as a cause of epileptic encephalopathy, which is characterized by refractory seizures with developmental delay and cognitive disability. We previously described the heterozygous SCN8Amissense mutation p.Asn1768Asp in a child with epileptic encephalopathy that included seizures, ataxia, and sudden unexpected death in epilepsy (SUDEP). The mutation results in increased persistent sodium current and hyperactivity of transfected neurons. We have characterized a knock-in mouse model expressing this dominant gain-of-function mutation to investigate the pathology of the altered channel in vivo. The mutant channel protein is stable in vivo. Heterozygous Scn8aN1768D/+ mice exhibit seizures and SUDEP, confirming the causality of the de novo mutation in the proband. Using video/EEG analysis, we detect ictal discharges that coincide with convulsive seizures and myoclonic jerks. Prior to seizure onset, heterozygous mutants are not defective in motor learning or fear conditioning, but do exhibit mild impairment of motor coordination and social discrimination. Homozygous mutant mice exhibit earlier seizure onset than heterozygotes and more rapid progression to death. Analysis of the intermediate phenotype of functionally hemizygous Scn8aN1768D/− mice indicates that severity is increased by a double dose of mutant protein and reduced by the presence of wild-type protein. Scn8aN1768D mutant mice provide a model of epileptic encephalopathy that will be valuable for studying the in vivo effects of hyperactive Nav1.6 and the response to therapeutic interventions.
Commentary Recent advances in genome editing have made it easier than ever to make animal models of human disease mutations. In many cases, genetic causes of epilepsy result from gain-offunction mutations that require site-specific knock-in of the mutation to model the human disorder. Traditionally, this was accomplished by homologous recombination in embryonic stem cells that could then be used to generate a mouse model. The efficiency of classical homologous recombination is extremely low, making this a labor-intensive and time-consuming process for model generation. The key discovery that programmable nucleases can target double-stranded breaks to specific sequences and enhance local DNA repair mechanisms paved the way for new genome editing technologies that are changing the landscape of animal model generation (1). There are several technologies that exploit this effect for site-specific genome editing, including zinc-finger nucleases (ZFNs), transcription activation-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs). There are pros and cons to each system that can guide the Epilepsy Currents, Vol. 15, No. 2 (March/April) 2015 pp. 83–84 © American Epilepsy Society
choice for specific applications (reviewed in [2]). TALENs have an extremely high success rate coupled with low potential for off-target effects and can be used to target virtually any DNA sequence. They have been successfully used to generate mice with targeted mutations by microinjecting the components directly into one-cell embryos, resulting in mutant mice within 2 months (3, 4). The speed and precision of TALENs make this an attractive approach for knocking-in human patient mutations. TALEN technology was recently utilized to generate a mouse model of SCN8A-associated epileptic encephalopathy (5). This new mouse model carries the Scn8a-N1768D mutation, which was identified as a dominant de novo mutation in a patient with epileptic encephalopathy (6). The proband carrying the SCN8A-N1768D mutation exhibited a syndrome that included early-onset seizures, intellectual disability, and features of autism, and ultimately succumbed to SUDEP in adolescence. Biophysical characterization of the mutation in a heterologous expression system demonstrated gain-offunction defects that were consistent with neuronal hyperexcitability (6). In the current study, Wagnon and colleagues evaluated mice carrying the Scn8a-N1768D mutation for a number of phenotypes, including lifespan, seizures, ataxia, motor and associative learning, and fear conditioning. In addition, the
83
SCN8A Epileptic Encephalopathy Mouse Model
authors evaluated mice with a number of different genotype combinations. Homozygous Scn8aN1768D/N1768D mice had a very severe phenotype with profound motor deficits and severe generalized tonic–clonic seizures that resulted in 100% lethality by 1 month of age. Heterozygous Scn8aN1768D/+ mice, which model the dominant condition observed in the patient, had a more complex phenotype that evolved over time. Initially, the Scn8aN1768D/+ mice appeared normal; however, they exhibited EEG abnormalities around 2 months of age, developed generalized tonic–clonic seizures at 3 months of age, and progressed to SUDEP within a month of seizure onset. To determine whether the more severe phenotype observed in the homozyogous Scn8aN1768D/N1768D mice was due to elevated dosage of the mutant allele or absence of the wild-type allele, the authors generated Scn8aN1768D/- mice that carried one N1768D allele and one null allele. The phenotype of these Scn8aN1768D/- mice was intermediate between the homozygous Scn8aN1768D/N1768D and heterozygous Scn8aN1768D/+ mice. This suggested that presence of the wild-type allele confers a protective effect in the heterozygous Scn8aN1768D/+ mice. In addition to evaluating the seizure and SUDEP phenotypes, the authors also examined performance of Scn8aN1768D/+ mice in some motor function and behavioral tasks. First, they looked at motor learning and coordination using an accelerating rotarod task, which requires the mice to maintain their balance on a rotating dowel as it accelerates. The Scn8aN1768D/+ mice exhibited motor learning, but their overall performance was impaired relative to wild-type controls. This result, combined with several other assessments of motor strength, indicated that Scn8aN1768D/+ mice have a deficit in motor coordination. To assess associative learning and memory, the authors used a fear conditioning paradigm. They did not observe any difference in performance between Scn8aN1768D/+ and wild-type mice. This indicated that the Scn8aN1768D/+ mice do not have an associative learning and memory deficit preceding seizure onset. It is possible that a deficit might emerge following the onset of spontaneous seizures. However, tests were only performed with young animals that had not yet experienced seizures. The authors also examined social interaction in the Scn8aN1768D/+ mice using a three-chambered social interaction test. In this test, mice are first given the opportunity to interact with a novel mouse or a novel inanimate object. In the next phase, mice are given the opportunity to interact with the original mouse, which is now familiar, or an unfamiliar mouse. Scn8aN1768D/+ mice showed normal sociability, preferring the novel mouse over an inanimate object. However, unlike wildtype mice, Scn8aN1768D/+ mice did not show a preference for the unfamiliar mouse relative to the familiar mouse. This suggests that they have a deficit in social discrimination. To rule out impaired olfaction as a potential cause for this deficit, they authors tested olfactory function and did not find an impairment. Together, these results imply that the social discrimination deficit could be due to impaired cognition. The authors are appropriately conservative in their interpretation of the behavioral testing results and conclude that additional testing is required to comprehensively evaluate cognitive function of Scn8aN1768D/+ mice.
84
The Scn8aN1768D+/- mouse model recapitulated many of the key features of human SCN8A-associated encephalopathy. Rodent genetic models can be fairly reliable for modeling some aspects of epileptic encephalopathy pathophysiology, including seizures and SUDEP (7–10). It is more challenging to determine if they recapitulate the behavioral and communication aspects of the human disorder. This requires using measures in these domains that are ethologically valid for rodents. In many ways, it is not surprising that dysfunction in more complex species-specific behaviors are difficult to model since the underlying networks are different in rodents and humans. Overall, rodent genetic models are very valuable for understanding the molecular mechanisms and physiologic consequences of epilepsy mutations. However, we should always bear in mind that animal models are not perfect, smaller copies of the human disorder but rather are a tractable starting point for generating and testing hypotheses about disease mechanisms and therapeutic strategies. by Jennifer A. Kearney, PhD References 1. Rouet P, Smih F, Jasin M. Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc Natl Acad Sci U S A 1994;91:6064–6068. 2. Kim H, Kim JS. A guide to genome engineering with programmable nucleases. Nat Rev Genet 2014;15:321–334. 3. Panda SK, Wefers B, Ortiz O, Floss T, Schmid B, Haass C, Wurst W, Kühn R. Highly efficient targeted mutagenesis in mice using TALENs. Genetics 2013;195:703–713. 4. Wefers B, Meyer M, Ortiz O, Hrabé de Angelis M, Hansen J, Wurst W, Kühn R. Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides. Proc Natl Acad Sci U S A 2013;110:3782–3787. 5. Jones JM, Meisler MH. Modeling human epilepsy by TALEN targeting of mouse sodium channel Scn8a. Genesis. 2014;52:141–148. 6. Veeramah KR, O’Brien JE, Meisler MH, Cheng X, Dib-Hajj SD, Waxman SG, Talwar D, Girirajan S, Eichler EE, Restifo LL, Erickson RP, Hammer MF. De novo pathogenic SCN8A mutation identified by whole-genome sequencing of a family quartet affected by infantile epileptic encephalopathy and SUDEP. Am J Hum Genet 2012;90:502–510. 7. Yu FH, Mantegazza M, Westenbroek RE, Robbins CA, Kalume F, Burton KA, Spain WJ, McKnight GS, Scheuer T, Catterall WA. Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat Neurosci 2006;9:1142–1149. 8. Ogiwara I, Miyamoto H, Morita N, Atapour N, Mazaki E, Inoue I, Takeuchi T, Itohara S, Yanagawa Y, Obata K, Furuichi T, Hensch TK, Yamakawa K. Nav1.1 localizes to axons of parvalbumin-positive inhibitory interneurons: A circuit basis for epileptic seizures in mice carrying an SCN1A gene mutation. J Neurosci 2007;27:5903–5914. 9. Price MG, Yoo JW, Burgess DL, Deng F, Hrachovy RA, Frost JD Jr, Noebels JL. A triplet repeat expansion genetic mouse model of infantile spasms syndrome, Arx(GCG)10+7, with interneuronopathy, spasms in infancy, persistent seizures, and adult cognitive and behavioral impairment. J Neurosci 2009;29:8752–8763. 10. Kehrl JM, Sahaya K, Dalton HM, Charbeneau RA, Kohut KT, Gilbert K, Pelz MC, Parent J, Neubig RR. Gain-of-function mutation in GNAO1: A murine model of epileptiform encephalopathy (EIEE17)? Mamm Genome 2014;25:202–210
TALE of an SCN8A-Associated Epileptic Encephalopathy Mouse Model
Convulsive Seizures and SUDEP in a Mouse Model of SCN8A Epileptic Encephalopathy. Wagnon JL, Korn MJ, Parent R, Tarpey TA, Jones JM, Hammer MF, Murphy GG, Parent JM, Meisler MH. [published online ahead of print September 26, 2014]. Human Molecular. pii: ddu470.
De novo mutations of the voltage-gated sodium channel gene SCN8A have recently been recognized as a cause of epileptic encephalopathy, which is characterized by refractory seizures with developmental delay and cognitive disability. We previously described the heterozygous SCN8Amissense mutation p.Asn1768Asp in a child with epileptic encephalopathy that included seizures, ataxia, and sudden unexpected death in epilepsy (SUDEP). The mutation results in increased persistent sodium current and hyperactivity of transfected neurons. We have characterized a knock-in mouse model expressing this dominant gain-of-function mutation to investigate the pathology of the altered channel in vivo. The mutant channel protein is stable in vivo. Heterozygous Scn8aN1768D/+ mice exhibit seizures and SUDEP, confirming the causality of the de novo mutation in the proband. Using video/EEG analysis, we detect ictal discharges that coincide with convulsive seizures and myoclonic jerks. Prior to seizure onset, heterozygous mutants are not defective in motor learning or fear conditioning, but do exhibit mild impairment of motor coordination and social discrimination. Homozygous mutant mice exhibit earlier seizure onset than heterozygotes and more rapid progression to death. Analysis of the intermediate phenotype of functionally hemizygous Scn8aN1768D/− mice indicates that severity is increased by a double dose of mutant protein and reduced by the presence of wild-type protein. Scn8aN1768D mutant mice provide a model of epileptic encephalopathy that will be valuable for studying the in vivo effects of hyperactive Nav1.6 and the response to therapeutic interventions.
Commentary Recent advances in genome editing have made it easier than ever to make animal models of human disease mutations. In many cases, genetic causes of epilepsy result from gain-offunction mutations that require site-specific knock-in of the mutation to model the human disorder. Traditionally, this was accomplished by homologous recombination in embryonic stem cells that could then be used to generate a mouse model. The efficiency of classical homologous recombination is extremely low, making this a labor-intensive and time-consuming process for model generation. The key discovery that programmable nucleases can target double-stranded breaks to specific sequences and enhance local DNA repair mechanisms paved the way for new genome editing technologies that are changing the landscape of animal model generation (1). There are several technologies that exploit this effect for site-specific genome editing, including zinc-finger nucleases (ZFNs), transcription activation-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs). There are pros and cons to each system that can guide the Epilepsy Currents, Vol. 15, No. 2 (March/April) 2015 pp. 83–84 © American Epilepsy Society
choice for specific applications (reviewed in [2]). TALENs have an extremely high success rate coupled with low potential for off-target effects and can be used to target virtually any DNA sequence. They have been successfully used to generate mice with targeted mutations by microinjecting the components directly into one-cell embryos, resulting in mutant mice within 2 months (3, 4). The speed and precision of TALENs make this an attractive approach for knocking-in human patient mutations. TALEN technology was recently utilized to generate a mouse model of SCN8A-associated epileptic encephalopathy (5). This new mouse model carries the Scn8a-N1768D mutation, which was identified as a dominant de novo mutation in a patient with epileptic encephalopathy (6). The proband carrying the SCN8A-N1768D mutation exhibited a syndrome that included early-onset seizures, intellectual disability, and features of autism, and ultimately succumbed to SUDEP in adolescence. Biophysical characterization of the mutation in a heterologous expression system demonstrated gain-offunction defects that were consistent with neuronal hyperexcitability (6). In the current study, Wagnon and colleagues evaluated mice carrying the Scn8a-N1768D mutation for a number of phenotypes, including lifespan, seizures, ataxia, motor and associative learning, and fear conditioning. In addition, the
83
SCN8A Epileptic Encephalopathy Mouse Model
authors evaluated mice with a number of different genotype combinations. Homozygous Scn8aN1768D/N1768D mice had a very severe phenotype with profound motor deficits and severe generalized tonic–clonic seizures that resulted in 100% lethality by 1 month of age. Heterozygous Scn8aN1768D/+ mice, which model the dominant condition observed in the patient, had a more complex phenotype that evolved over time. Initially, the Scn8aN1768D/+ mice appeared normal; however, they exhibited EEG abnormalities around 2 months of age, developed generalized tonic–clonic seizures at 3 months of age, and progressed to SUDEP within a month of seizure onset. To determine whether the more severe phenotype observed in the homozyogous Scn8aN1768D/N1768D mice was due to elevated dosage of the mutant allele or absence of the wild-type allele, the authors generated Scn8aN1768D/- mice that carried one N1768D allele and one null allele. The phenotype of these Scn8aN1768D/- mice was intermediate between the homozygous Scn8aN1768D/N1768D and heterozygous Scn8aN1768D/+ mice. This suggested that presence of the wild-type allele confers a protective effect in the heterozygous Scn8aN1768D/+ mice. In addition to evaluating the seizure and SUDEP phenotypes, the authors also examined performance of Scn8aN1768D/+ mice in some motor function and behavioral tasks. First, they looked at motor learning and coordination using an accelerating rotarod task, which requires the mice to maintain their balance on a rotating dowel as it accelerates. The Scn8aN1768D/+ mice exhibited motor learning, but their overall performance was impaired relative to wild-type controls. This result, combined with several other assessments of motor strength, indicated that Scn8aN1768D/+ mice have a deficit in motor coordination. To assess associative learning and memory, the authors used a fear conditioning paradigm. They did not observe any difference in performance between Scn8aN1768D/+ and wild-type mice. This indicated that the Scn8aN1768D/+ mice do not have an associative learning and memory deficit preceding seizure onset. It is possible that a deficit might emerge following the onset of spontaneous seizures. However, tests were only performed with young animals that had not yet experienced seizures. The authors also examined social interaction in the Scn8aN1768D/+ mice using a three-chambered social interaction test. In this test, mice are first given the opportunity to interact with a novel mouse or a novel inanimate object. In the next phase, mice are given the opportunity to interact with the original mouse, which is now familiar, or an unfamiliar mouse. Scn8aN1768D/+ mice showed normal sociability, preferring the novel mouse over an inanimate object. However, unlike wildtype mice, Scn8aN1768D/+ mice did not show a preference for the unfamiliar mouse relative to the familiar mouse. This suggests that they have a deficit in social discrimination. To rule out impaired olfaction as a potential cause for this deficit, they authors tested olfactory function and did not find an impairment. Together, these results imply that the social discrimination deficit could be due to impaired cognition. The authors are appropriately conservative in their interpretation of the behavioral testing results and conclude that additional testing is required to comprehensively evaluate cognitive function of Scn8aN1768D/+ mice.
84
The Scn8aN1768D+/- mouse model recapitulated many of the key features of human SCN8A-associated encephalopathy. Rodent genetic models can be fairly reliable for modeling some aspects of epileptic encephalopathy pathophysiology, including seizures and SUDEP (7–10). It is more challenging to determine if they recapitulate the behavioral and communication aspects of the human disorder. This requires using measures in these domains that are ethologically valid for rodents. In many ways, it is not surprising that dysfunction in more complex species-specific behaviors are difficult to model since the underlying networks are different in rodents and humans. Overall, rodent genetic models are very valuable for understanding the molecular mechanisms and physiologic consequences of epilepsy mutations. However, we should always bear in mind that animal models are not perfect, smaller copies of the human disorder but rather are a tractable starting point for generating and testing hypotheses about disease mechanisms and therapeutic strategies. by Jennifer A. Kearney, PhD References 1. Rouet P, Smih F, Jasin M. Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc Natl Acad Sci U S A 1994;91:6064–6068. 2. Kim H, Kim JS. A guide to genome engineering with programmable nucleases. Nat Rev Genet 2014;15:321–334. 3. Panda SK, Wefers B, Ortiz O, Floss T, Schmid B, Haass C, Wurst W, Kühn R. Highly efficient targeted mutagenesis in mice using TALENs. Genetics 2013;195:703–713. 4. Wefers B, Meyer M, Ortiz O, Hrabé de Angelis M, Hansen J, Wurst W, Kühn R. Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides. Proc Natl Acad Sci U S A 2013;110:3782–3787. 5. Jones JM, Meisler MH. Modeling human epilepsy by TALEN targeting of mouse sodium channel Scn8a. Genesis. 2014;52:141–148. 6. Veeramah KR, O’Brien JE, Meisler MH, Cheng X, Dib-Hajj SD, Waxman SG, Talwar D, Girirajan S, Eichler EE, Restifo LL, Erickson RP, Hammer MF. De novo pathogenic SCN8A mutation identified by whole-genome sequencing of a family quartet affected by infantile epileptic encephalopathy and SUDEP. Am J Hum Genet 2012;90:502–510. 7. Yu FH, Mantegazza M, Westenbroek RE, Robbins CA, Kalume F, Burton KA, Spain WJ, McKnight GS, Scheuer T, Catterall WA. Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat Neurosci 2006;9:1142–1149. 8. Ogiwara I, Miyamoto H, Morita N, Atapour N, Mazaki E, Inoue I, Takeuchi T, Itohara S, Yanagawa Y, Obata K, Furuichi T, Hensch TK, Yamakawa K. Nav1.1 localizes to axons of parvalbumin-positive inhibitory interneurons: A circuit basis for epileptic seizures in mice carrying an SCN1A gene mutation. J Neurosci 2007;27:5903–5914. 9. Price MG, Yoo JW, Burgess DL, Deng F, Hrachovy RA, Frost JD Jr, Noebels JL. A triplet repeat expansion genetic mouse model of infantile spasms syndrome, Arx(GCG)10+7, with interneuronopathy, spasms in infancy, persistent seizures, and adult cognitive and behavioral impairment. J Neurosci 2009;29:8752–8763. 10. Kehrl JM, Sahaya K, Dalton HM, Charbeneau RA, Kohut KT, Gilbert K, Pelz MC, Parent J, Neubig RR. Gain-of-function mutation in GNAO1: A murine model of epileptiform encephalopathy (EIEE17)? Mamm Genome 2014;25:202–210
