Dravet syndrome is a severe, childhood-onset epilepsy largely due to heterozygous loss-of-function mutation of the gene
, which encodes the type 1 neuronal voltage-gated sodium (Na
) channel α ...subunit Nav1.1. Prior studies in mouse models of Dravet syndrome (
mice) indicate that, in cerebral cortex, Nav1.1 is predominantly expressed in GABAergic interneurons, in particular in parvalbumin-positive fast-spiking basket cell interneurons (PVINs). This has led to a model of Dravet syndrome pathogenesis in which Nav1.1 mutation leads to preferential dysfunction of interneurons, decreased synaptic inhibition, hyperexcitability, and epilepsy. However, such studies have been implemented at early developmental time points. Here, we performed electrophysiological recordings in acute brain slices prepared from male and female
mice as well as age-matched wild-type littermate controls and found that, later in development, the excitability of PVINs had normalized. Analysis of action potential waveforms indirectly suggests a reorganization of axonal Na
channels in PVINs from
mice, a finding supported by immunohistochemical data showing elongation of the axon initial segment. Our results imply that transient impairment of action potential generation by PVINs may contribute to the initial appearance of epilepsy, but is not the mechanism of ongoing, chronic epilepsy in Dravet syndrome.
Dravet syndrome is characterized by normal early development, temperature-sensitive seizures in infancy, progression to treatment-resistant epilepsy, developmental delay, autism, and sudden unexplained death due to mutation in
encoding the Na+ channel subunit Nav1.1. Prior work has revealed a preferential impact of Nav1.1 loss on the function of GABAergic inhibitory interneurons. However, such data derive exclusively from recordings of neurons in young
mice. Here, we show that impaired action potential generation observed in parvalbumin-positive fast-spiking interneurons (PVINs) in
+/- mice during early development has normalized by postnatal day 35. This work suggests that a transient impairment of PVINs contributes to epilepsy onset, but is not the mechanism of ongoing, chronic epilepsy in Dravet syndrome.
A substantial number of mutations have been identified in voltage-gated sodium channel genes that result in various forms of human epilepsy. SCN1A mutations result in a spectrum of severity ranging ...from mild febrile seizures to Dravet syndrome, an infant-onset epileptic encephalopathy. Dravet syndrome patients experience multiple seizures types that are often refractory to treatment, developmental delays, and elevated risk for SUDEP. The same sodium channel mutation can produce epilepsy phenotypes of varying clinical severity. This suggests that other factors, including genetic, modify the primary mutation and change disease severity. Mouse models provide a useful tool in studying the genetic basis of epilepsy. The mouse strain background can alter phenotype severity, supporting a contribution of genetic modifiers in epilepsy. The Scn1a+/- mouse model has a strain-dependent epilepsy phenotype. Scn1a+/- mice on the 129S6/SvEvTac (129) strain have a normal phenotype and lifespan, while 129xC57BL/6JF1-Scn1a+/- mice experience spontaneous seizures, hyperthermia-induced seizures and high rates of premature death. We hypothesize the phenotypic differences are due to strain-specific genetic modifiers that influence expressivity of the Scn1a+/- phenotype. Low resolution mapping of Scn1a+/- identified several Dravet syndrome modifier (Dsm) loci responsible for the strain-dependent difference in survival. One locus of interest, Dsm1 located on chromosome 5, was fine mapped to a 9 Mb region using interval specific congenics. RNA-Seq was then utilized to identify candidate modifier genes within this narrowed region. Three genes with significant total gene expression differences between 129S6/SvEvTac and 129xC57BL/6JF1 were identified, including the GABAA receptor subunit, Gabra2. Further analysis of Gabra2 demonstrated allele-specific expression. Pharmological manipulation by clobazam, a common anticonvulsant with preferential affinity for the GABRA2 receptor, revealed dose-dependent protection against hyperthermia-induced seizures in Scn1a+/- mice. These findings support Gabra2 as a genetic modifier of the Scn1a+/- mouse model of Dravet syndrome.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Hill-type muscle models are widely employed in simulations of human movement. Yet, the parameters underlying these models are difficult or impossible to measure in vivo. Prior studies demonstrate ...that Hill-type muscle parameters are encoded within dynamometric data. But, a generalizable approach for estimating these parameters from dynamometric data has not been realized. We aimed to leverage musculoskeletal models and artificial neural networks to classify one Hill-type muscle parameter (maximum isometric force) from easily measurable dynamometric data (simulated lateral pinch force). We tested two neural networks (feedforward and long short-term memory) to identify if accounting for dynamic behavior improved accuracy.
We generated four datasets via forward dynamics, each with increasing complexity from adjustments to more muscles. Simulations were grouped and evaluated to show how varying the maximum isometric force of thumb muscles affects lateral pinch force. Both neural networks classified these groups from lateral pinch force alone.
Both neural networks achieved accuracies above 80% for datasets which varied only the flexor pollicis longus and/or the abductor pollicis longus. The inclusion of muscles with redundant functions dropped model accuracies to below 30%. While both neural networks were consistently more accurate than random guess, the long short-term memory model was not consistently more accurate than the feedforward model.
Our investigations demonstrate that artificial neural networks provide an inexpensive, data-driven approach for approximating Hill-type muscle-tendon parameters from easily measurable data. However, muscles of redundant function or of little impact to force production make parameter classification more challenging.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Objective
Numerous studies have demonstrated increased load of de novo copy number variants or single nucleotide variants in individuals with neurodevelopmental disorders, including epileptic ...encephalopathies, intellectual disability, and autism.
Methods
We searched for de novo mutations in a family quartet with a sporadic case of epileptic encephalopathy with no known etiology to determine the underlying cause using high‐coverage whole exome sequencing (WES) and lower‐coverage whole genome sequencing. Mutations in additional patients were identified by WES. The effect of mutations on protein function was assessed in a heterologous expression system.
Results
We identified a de novo missense mutation in KCNB1 that encodes the KV2.1 voltage‐gated potassium channel. Functional studies demonstrated a deleterious effect of the mutation on KV2.1 function leading to a loss of ion selectivity and gain of a depolarizing inward cation conductance. Subsequently, we identified 2 additional patients with epileptic encephalopathy and de novo KCNB1 missense mutations that cause a similar pattern of KV2.1 dysfunction.
Interpretation
Our genetic and functional evidence demonstrate that KCNB1 mutation can result in early onset epileptic encephalopathy. This expands the locus heterogeneity associated with epileptic encephalopathies and suggests that clinical WES may be useful for diagnosis of epileptic encephalopathies of unknown etiology. Ann Neurol 2014;76:529–540
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Dravet syndrome is a developmental and epileptic encephalopathy (DEE) characterized by intractable seizures, comorbidities related to developmental, cognitive, and motor delays, and a high mortality ...burden due to sudden unexpected death in epilepsy (SUDEP). Most Dravet syndrome cases are attributed to
SCN1A
haploinsufficiency, with genetic modifiers and environmental factors influencing disease severity. Mouse models with heterozygous deletion of
Scn1a
recapitulate key features of Dravet syndrome, including seizures and premature mortality; however, severity varies depending on genetic background. Here, we refined two Dravet survival modifier (
Dsm
) loci,
Dsm2
on chromosome 7 and
Dsm3
on chromosome 8, using interval-specific congenic (ISC) mapping.
Dsm2
was complex and encompassed at least two separate loci, while
Dsm3
was refined to a single locus. Candidate modifier genes within these refined loci were prioritized based on brain expression, strain-dependent differences, and biological relevance to seizures or epilepsy. High priority candidate genes for
Dsm2
include
Nav2
,
Ptpn5
,
Ldha
,
Dbx1
,
Prmt3
and
Slc6a5
, while
Dsm3
has a single high priority candidate,
Psd3
. This study underscores the complex genetic architecture underlying Dravet syndrome and provides insights into potential modifier genes that could influence disease severity and serve as novel therapeutic targets.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Abstract Heterozygous loss-of-function SCN1A mutations cause Dravet syndrome, an epileptic encephalopathy of infancy that exhibits variable clinical severity. We utilized a heterozygous Scn1a ...knockout ( Scn1a+/− ) mouse model of Dravet syndrome to investigate the basis for phenotype variability. These animals exhibit strain-dependent seizure severity and survival. Scn1a+/− mice on strain 129S6/SvEvTac (129. Scn1a+/− ) have no overt phenotype and normal survival compared with Scn1a+/− mice bred to C57BL/6J (F1. Scn1a+/− ) that have severe epilepsy and premature lethality. We tested the hypothesis that strain differences in sodium current (INa ) density in hippocampal neurons contribute to these divergent phenotypes. Whole-cell voltage-clamp recording was performed on acutely-dissociated hippocampal neurons from postnatal days 21–24 (P21–24) 129. Scn1a+/− or F1. Scn1a+/− mice and wild-type littermates. INa density was lower in GABAergic interneurons from F1. Scn1a+/− mice compared to wild-type littermates, while on the 129 strain there was no difference in GABAergic interneuron INa density between 129. Scn1a+/− mice and wild-type littermate controls. By contrast, INa density was elevated in pyramidal neurons from both 129. Scn1a+/− and F1. Scn1a+/− mice, and was correlated with more frequent spontaneous action potential firing in these neurons, as well as more sustained firing in F1. Scn1a+/− neurons. We also observed age-dependent differences in pyramidal neuron INa density between wild-type and Scn1a+/− animals. We conclude that preserved INa density in GABAergic interneurons contributes to the milder phenotype of 129. Scn1a+/− mice. Furthermore, elevated INa density in excitatory pyramidal neurons at P21–24 correlates with age-dependent onset of lethality in F1. Scn1a+/− mice. Our findings illustrate differences in hippocampal neurons that may underlie strain- and age-dependent phenotype severity in a Dravet syndrome mouse model, and emphasize a contribution of pyramidal neuron excitability.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Summary
Dravet syndrome, an early onset epileptic encephalopathy, is most often caused by de novo mutation of the neuronal voltage‐gated sodium channel gene SCN1A. Mouse models with deletion of Scn1a ...recapitulate Dravet syndrome phenotypes, including spontaneous generalized tonic–clonic seizures, susceptibility to seizures induced by elevated body temperature, and elevated risk of sudden unexpected death in epilepsy. Importantly, the epilepsy phenotype of Dravet mouse models is highly strain‐dependent, suggesting a strong influence of genetic modifiers. We previously identified Cacna1g, encoding the Cav3.1 subunit of the T‐type calcium channel family, as an epilepsy modifier in the Scn2aQ54 transgenic epilepsy mouse model. In this study, we asked whether transgenic alteration of Cacna1g expression modifies severity of the Scn1a+/− Dravet phenotype. Scn1a+/− mice with decreased Cacna1g expression showed partial amelioration of disease phenotypes with improved survival and reduced spontaneous seizure frequency. However, reduced Cacna1g expression did not alter susceptibility to hyperthermia‐induced seizures. Transgenic elevation of Cacna1g expression had no effect on the Scn1a+/− epilepsy phenotype. These results provide support for Cacna1g as a genetic modifier in a mouse model of Dravet syndrome and suggest that Cav3.1 may be a potential molecular target for therapeutic intervention in patients.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
KCNT1 encodes the sodium-activated potassium channel KNa1.1, expressed preferentially in the frontal cortex, hippocampus, cerebellum, and brainstem. Pathogenic missense variants in KCNT1 are ...associated with intractable epilepsy, namely epilepsy of infancy with migrating focal seizures (EIMFS), and sleep-related hypermotor epilepsy (SHE). In vitro studies of pathogenic KCNT1 variants support predominantly a gain-of-function molecular mechanism, but how these variants behave in a neuron or ultimately drive formation of an epileptogenic circuit is an important and timely question. Using CRISPR/Cas9 gene editing, we introduced a gain-of-function variant into the endogenous mouse Kcnt1 gene. Compared to wild-type (WT) littermates, heterozygous and homozygous knock-in mice displayed greater seizure susceptibility to the chemoconvulsants kainate and pentylenetetrazole (PTZ), but not to flurothyl. Using acute slice electrophysiology in heterozygous and homozygous Kcnt1 knock-in and WT littermates, we demonstrated that CA1 hippocampal pyramidal neurons exhibit greater amplitude of miniature inhibitory postsynaptic currents in mutant mice with no difference in frequency, suggesting greater inhibitory tone associated with the Kcnt1 mutation. To address alterations in GABAergic signaling, we bred Kcnt1 knock-in mice to a parvalbumin-tdTomato reporter line, and found that parvalbumin-expressing (PV+) interneurons failed to fire repetitively with large amplitude current injections and were more prone to depolarization block. These alterations in firing can be recapitulated by direct application of the KNa1.1 channel activator loxapine in WT but are occluded in knock-in littermates, supporting a direct channel gain-of-function mechanism. Taken together, these results suggest that KNa1.1 gain-of-function dampens interneuron excitability to a greater extent than it impacts pyramidal neuron excitability, driving seizure susceptibility in a mouse model of KCNT1-associated epilepsy.
•Kcnt1 RNA and protein are expressed in parvalbumin-positive interneurons.•Mice with a Kcnt1 gain-of-function variant (Kcnt1-L437F) have greater seizure susceptibility•Parvalbumin-positive interneurons from Kcnt1-L437F mice exhibit impaired ability to fire repetitively and are prone to depolarization block.•Loxapine phenocopies cell-specific alterations of high-frequency firing in wildtype neurons.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract Mutations in voltage-gated sodium channels expressed highly in the brain ( SCN1A , SCN2A , SCN3A , and SCN8A ) are responsible for an increasing number of epilepsy syndromes. In particular, ...mutations in the SCN3A gene, encoding the pore-forming Nav 1.3 α subunit, have been identified in patients with focal epilepsy. Biophysical characterization of epilepsy-associated SCN3A variants suggests that both gain- and loss-of-function SCN3A mutations may lead to increased seizure susceptibility. In this report, we identified a novel SCN3A variant (L247P) by whole exome sequencing of a child with focal epilepsy, developmental delay, and autonomic nervous system dysfunction. Voltage clamp analysis showed no detectable sodium current in a heterologous expression system expressing the SCN3A-L247P variant. Furthermore, cell surface biotinylation demonstrated a reduction in the amount of SCN3A-L247P at the cell surface, suggesting the SCN3A-L247P variant is a trafficking-deficient mutant. To further explore the possible clinical consequences of reduced SCN3A activity, we investigated the effect of a hypomorphic Scn3a allele ( Scn3a Hyp ) on seizure susceptibility and behavior using a gene trap mouse line. Heterozygous Scn3a mutant mice ( Scn3a +/ Hyp ) did not exhibit spontaneous seizures nor were they susceptible to hyperthermia-induced seizures. However, they displayed increased susceptibility to electroconvulsive (6 Hz) and chemiconvulsive (flurothyl and kainic acid) induced seizures. Scn3a +/ Hyp mice also exhibited deficits in locomotor activity and motor learning. Taken together, these results provide evidence that loss-of-function of SCN3A caused by reduced protein expression or deficient trafficking to the plasma membrane may contribute to increased seizure susceptibility.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
A purified preparation of cannabidiol (CBD), a cannabis constituent, has been approved for the treatment of intractable childhood epilepsies such as Dravet syndrome. Extensive pharmacological ...characterization of CBD shows activity at numerous molecular targets but its anticonvulsant mechanism(s) of action is yet to be delineated. Many suggest that the anticonvulsant action of CBD is the result of G protein-coupled receptor 55 (GPR55) inhibition. Here we assessed whether Gpr55 contributes to the strain-dependent seizure phenotypes of the Scn1a+/- mouse model of Dravet syndrome. The Scn1a+/- mice on a 129S6/SvEvTac (129) genetic background have no overt phenotype, while those on a 129 x C57BL/6J F1 background exhibit a severe phenotype that includes hyperthermia-induced seizures, spontaneous seizures and reduced survival. We observed greater Gpr55 transcript expression in the cortex and hippocampus of mice on the seizure-susceptible F1 background compared to those on the seizure-resistant 129 genetic background, suggesting that Gpr55 might be a genetic modifier of Scn1a+/- mice. We examined the effect of heterozygous genetic deletion of Gpr55 and pharmacological inhibition of GPR55 on the seizure phenotypes of F1.Scn1a+/- mice. Heterozygous Gpr55 deletion and inhibition of GPR55 with CID2921524 did not affect the temperature threshold of a thermally-induced seizure in F1.Scn1a+/- mice. Neither was there an effect of heterozygous Gpr55 deletion observed on spontaneous seizure frequency or survival of F1.Scn1a+/- mice. Our results suggest that GPR55 antagonism may not be a suitable anticonvulsant target for Dravet syndrome drug development programs, although future research is needed to provide more definitive conclusions.
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