ABSTRACT
Genetic engineering techniques have contributed to the now widespread use of zebrafish to investigate gene function, but zebrafish-based human disease studies, and particularly for neurological disorders, are limited. Here we used CRISPR-Cas9 to generate 40 single-gene mutant zebrafish lines representing catastrophic childhood epilepsies. We evaluated larval phenotypes using electrophysiological, behavioral, neuro-anatomical, survival and pharmacological assays. Local field potential recordings (LFP) were used to screen â¼3300 larvae. Phenotypes with unprovoked electrographic seizure activity (i.e., epilepsy) were identified in zebrafish lines for 8 genes; ARX, EEF1A, GABRB3, GRIN1, PNPO, SCN1A, STRADA and STXBP1. We also created an open-source database containing sequencing information, survival curves, behavioral profiles and representative electrophysiology data. We offer all zebrafish lines as a resource to the neuroscience community and envision them as a starting point for further functional analysis and/or identification of new therapies.
Subject(s)
Disease Models, Animal , Embryo, Nonmammalian/metabolism , Epilepsy/genetics , Zebrafish Proteins/genetics , Zebrafish/genetics , Animals , Animals, Genetically Modified , Child , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/embryology , Epilepsy/pathology , Epilepsy/physiopathology , Gene Expression , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Larva/genetics , Mutation , Phenotype , Survival Analysis , Exome Sequencing/methods , Zebrafish/embryologyABSTRACT
Energy-producing pathways are novel therapeutic targets for the treatment of neurodevelopmental disorders. Here, we focussed on correcting metabolic defects in a catastrophic paediatric epilepsy, Dravet syndrome which is caused by mutations in sodium channel NaV1.1 gene, SCN1A. We utilized a translatable zebrafish model of Dravet syndrome (scn1lab) which exhibits key characteristics of patients with Dravet syndrome and shows metabolic deficits accompanied by down-regulation of gluconeogenesis genes, pck1 and pck2. Using a metabolism-based small library screen, we identified compounds that increased gluconeogenesis via up-regulation of pck1 gene expression in scn1lab larvae. Treatment with PK11195, a pck1 activator and a translocator protein ligand, normalized dys-regulated glucose levels, metabolic deficits, translocator protein expression and significantly decreased electrographic seizures in mutant larvae. Inhibition of pck1 in wild-type larvae mimicked metabolic and behaviour defects observed in scn1lab mutants. Together, this suggests that correcting dys-regulated metabolic pathways can be therapeutic in neurodevelopmental disorders such as Dravet syndrome arising from ion channel dysfunction.
ABSTRACT
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ABSTRACT
Hemizygous mutations in the human gene encoding platelet-activating factor acetylhydrolase IB subunit alpha (Pafah1b1), also called Lissencephaly-1, can cause classical lissencephaly, a severe malformation of cortical development. Children with this disorder suffer from deficits in neuronal migration, severe intellectual disability, intractable epilepsy and early death. While many of these features can be reproduced in Pafah1b1+/- mice, the impact of Pafah1b1+/- on the function of individual subpopulations of neurons and ultimately brain circuits is largely unknown. Here, we show tangential migration of young GABAergic interneurons into the developing hippocampus is slowed in Pafah1b1+/- mice. Mutant mice had a decreased density of parvalbumin- and somatostatin-positive interneurons in dentate gyrus, but no change in density of calretinin interneurons. Whole-cell patch-clamp recordings revealed increased excitatory and decreased inhibitory synaptic inputs onto granule cells of Pafah1b1+/- mice. Mutant animals developed spontaneous electrographic seizures, as well as long-term deficits in contextual memory. Our findings provide evidence of a dramatic shift in excitability in the dentate gyrus of Pafah1b1+/- mice that may contribute to epilepsy or cognitive impairments associated with lissencephaly.
ABSTRACT
Altered metabolism is an important feature of many epileptic syndromes but has not been reported in Dravet syndrome (DS), a catastrophic childhood epilepsy associated with mutations in a voltage-activated sodium channel, Nav1.1 (SCN1A). To address this, we developed novel methodology to assess real-time changes in bioenergetics in zebrafish larvae between 4 and 6 d postfertilization (dpf). Baseline and 4-aminopyridine (4-AP) stimulated glycolytic flux and mitochondrial respiration were simultaneously assessed using a Seahorse Biosciences extracellular flux analyzer. Scn1Lab mutant zebrafish showed a decrease in baseline glycolytic rate and oxygen consumption rate (OCR) compared to controls. A ketogenic diet formulation rescued mutant zebrafish metabolism to control levels. Increasing neuronal excitability with 4-AP resulted in an immediate increase in glycolytic rates in wild-type zebrafish, whereas mitochondrial OCR increased slightly and quickly recovered to baseline values. In contrast, scn1Lab mutant zebrafish showed a significantly slower and exaggerated increase of both glycolytic rates and OCR after 4-AP. The underlying mechanism of decreased baseline OCR in scn1Lab mutants was not because of altered mitochondrial DNA content or dysfunction of enzymes in the electron transport chain or tricarboxylic acid cycle. Examination of glucose metabolism using a PCR array identified five glycolytic genes that were downregulated in scn1Lab mutant zebrafish. Our findings in scn1Lab mutant zebrafish suggest that glucose and mitochondrial hypometabolism contribute to the pathophysiology of DS.
Subject(s)
Epilepsies, Myoclonic/physiopathology , Glycolysis/genetics , Mitochondria/metabolism , Oxygen Consumption/genetics , 4-Aminopyridine/pharmacology , Animals , Animals, Genetically Modified , Citric Acid Cycle/drug effects , Citric Acid Cycle/genetics , Diet, Ketogenic/methods , Disease Models, Animal , Epilepsies, Myoclonic/diet therapy , Epilepsies, Myoclonic/genetics , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Glycolysis/drug effects , Histocompatibility Antigens/metabolism , Larva , Mitochondria/drug effects , Mutation/genetics , NAV1.1 Voltage-Gated Sodium Channel/genetics , NAV1.1 Voltage-Gated Sodium Channel/metabolism , Oxygen Consumption/drug effects , Potassium Channel Blockers/pharmacology , Statistics, Nonparametric , ZebrafishABSTRACT
Mutations in a voltage-gated sodium channel (SCN1A) result in Dravet Syndrome (DS), a catastrophic childhood epilepsy. Zebrafish with a mutation in scn1Lab recapitulate salient phenotypes associated with DS, including seizures, early fatality, and resistance to antiepileptic drugs. To discover new drug candidates for the treatment of DS, we screened a chemical library of â¼1000 compounds and identified 4 compounds that rescued the behavioral seizure component, including 1 compound (dimethadione) that suppressed associated electrographic seizure activity. Fenfluramine, but not huperzine A, also showed antiepileptic activity in our zebrafish assays. The effectiveness of compounds that block neuronal calcium current (dimethadione) or enhance serotonin signaling (fenfluramine) in our zebrafish model suggests that these may be important therapeutic targets in patients with DS. Over 150 compounds resulting in fatality were also identified. We conclude that the combination of behavioral and electrophysiological assays provide a convenient, sensitive, and rapid basis for phenotype-based drug screening in zebrafish mimicking a genetic form of epilepsy.
ABSTRACT
Most cortical interneurons originate in a region of the embryonic subpallium called the medial ganglionic eminence (MGE). When MGE cells are transplanted into cerebral cortex, these progenitors migrate extensively and differentiate into functional inhibitory neurons. Although MGE progenitors have therapeutic potential following transplantation, it is unknown precisely how these cells distribute within neocortical lamina of the recipient brain. Here we transplanted mouse embryonic day 12.5 MGE progenitors into postnatal neocortex and evaluated laminar distribution of interneuron subtypes using double- and triple-label immunohistochemistry. Studies were performed using wild type (WT) or donor mice lacking a metabotropic GABA(B) receptor subunit (GABA(B1)R KO). MGE-derived neurons from WT and GABA(B1)R KO mice preferentially and densely distributed in neocortical layers 2/3, 5 and 6. As expected, MGE-derived neurons differentiated into parvalbumin+ and somatostatin+ interneurons within these neocortical lamina. Our findings provide insights into the anatomical integration of MGE-derived interneurons following transplantation.
Subject(s)
Embryonic Stem Cells/transplantation , GABAergic Neurons/cytology , Interneurons/cytology , Neocortex/cytology , Neural Stem Cells/transplantation , Receptors, GABA-B/metabolism , Animals , Cell Count , GABAergic Neurons/metabolism , Interneurons/metabolism , Median Eminence/cytology , Mice, Knockout , Neocortex/metabolism , Receptors, GABA-B/geneticsABSTRACT
Dravet syndrome is a catastrophic pediatric epilepsy with severe intellectual disability, impaired social development and persistent drug-resistant seizures. One of its primary monogenic causes are mutations in Nav1.1 (SCN1A), a voltage-gated sodium channel. Here we characterize zebrafish Nav1.1 (scn1Lab) mutants originally identified in a chemical mutagenesis screen. Mutants exhibit spontaneous abnormal electrographic activity, hyperactivity and convulsive behaviours. Although scn1Lab expression is reduced, microarray analysis is remarkable for the small fraction of differentially expressed genes (~3%) and lack of compensatory expression changes in other scn subunits. Ketogenic diet, diazepam, valproate, potassium bromide and stiripentol attenuate mutant seizure activity; seven other antiepileptic drugs have no effect. A phenotype-based screen of 320 compounds identifies a US Food and Drug Administration-approved compound (clemizole) that inhibits convulsive behaviours and electrographic seizures. This approach represents a new direction in modelling pediatric epilepsy and could be used to identify novel therapeutics for any monogenic epilepsy disorder.
Subject(s)
Anticonvulsants/therapeutic use , Benzimidazoles/therapeutic use , Drug Evaluation, Preclinical , NAV1.1 Voltage-Gated Sodium Channel/metabolism , Animals , Anticonvulsants/pharmacology , Benzimidazoles/pharmacology , Bromides/pharmacology , Diazepam/pharmacology , Dioxolanes/pharmacology , Epilepsies, Myoclonic/drug therapy , Gene Expression Profiling , Mutation , NAV1.1 Voltage-Gated Sodium Channel/genetics , Potassium Compounds/pharmacology , Seizures/drug therapy , Seizures/genetics , Valproic Acid/pharmacology , Voltage-Gated Sodium Channel beta-1 Subunit/genetics , Voltage-Gated Sodium Channel beta-1 Subunit/metabolism , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Type I lissencephaly, a neuronal migration disorder characterized by cognitive disability and refractory epilepsy, is often caused by heterozygous mutations in the LIS1 gene. Histopathologies of malformation-associated epilepsies have been well described, but it remains unclear whether hyperexcitability is attributable to disruptions in neuronal organization or abnormal circuit function. Here, we examined the effect of LIS1 deficiency on excitatory synaptic function in the dentate gyrus of hippocampus, a region believed to serve critical roles in seizure generation and learning and memory. Mice with heterozygous deletion of LIS1 exhibited robust granule cell layer dispersion, and adult-born granule cells labeled with enhanced green fluorescent protein were abnormally positioned in the molecular layer, hilus, and granule cell layer. In whole-cell patch-clamp recordings, reduced LIS1 function was associated with greater excitatory synaptic input to mature granule cells that was consistent with enhanced release probability at glutamatergic synapses. Adult-born granule cells that were ectopically positioned in the molecular layer displayed a more rapid functional maturation and integration into the synaptic network compared with newborn granule cells located in the hilus or granule cell layer or in wild-type controls. In a conditional knock-out mouse, induced LIS1 deficiency in adulthood also enhanced the excitatory input to granule cells in the absence of neuronal disorganization. These findings indicate that disruption of LIS1 has direct effects on excitatory synaptic transmission independent of laminar disorganization, and the ectopic position of adult-born granule cells within a malformed dentate gyrus critically influences their functional maturation and integration.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Aging , Cerebellar Nuclei/physiopathology , Classical Lissencephalies and Subcortical Band Heterotopias/physiopathology , Microtubule-Associated Proteins/metabolism , Neurons , Synapses , 1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , Animals , Mice , Mice, Knockout , Mice, Transgenic , Microtubule-Associated Proteins/genetics , Synaptic TransmissionABSTRACT
Disruption of E3 ubiquitin ligase activity in immature zebrafish mind bomb mutants leads to a failure in Notch signaling, excessive numbers of neurons, and depletion of neural progenitor cells. This neurogenic phenotype is associated with defects in neural patterning and brain development. Because developmental brain abnormalities are recognized as an important feature of childhood neurological disorders such as epilepsy and autism, we determined whether zebrafish mutants with grossly abnormal brain structure exhibit spontaneous electrical activity that resembles the long-duration, high-amplitude multispike discharges reported in immature zebrafish exposed to convulsant drugs. Electrophysiological recordings from agar immobilized mind bomb mutants at 3 d postfertilization confirmed the occurrence of electrographic seizure activity; seizure-like behaviors were also noted during locomotion video tracking of freely behaving mutants. To identify genes differentially expressed in the mind bomb mutant and provide insight into molecular pathways that may mediate these epileptic phenotypes, a transcriptome analysis was performed using microarray. Interesting candidate genes were further analyzed using conventional reverse transcriptase-PCR and real-time quantitative PCR, as well as whole-mount in situ hybridization. Approximately 150 genes, some implicated in development, transcription, cell metabolism, and signal transduction, are differentially regulated, including downregulation of several genes necessary for GABA-mediated signaling. These findings identify a collection of gene transcripts that may be responsible for the abnormal electrical discharge and epileptic activities observed in a mind bomb zebrafish mutant. This work may have important implications for neurological and neurodevelopmental disorders associated with mutations in ubiquitin ligase activity.
Subject(s)
Brain/metabolism , Neurons/metabolism , Seizures/genetics , Signal Transduction/genetics , Ubiquitin-Protein Ligases/metabolism , Zebrafish Proteins/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Brain/physiopathology , Electrophysiology , Female , In Situ Hybridization , Male , Motor Activity/genetics , Oligonucleotide Array Sequence Analysis , Phenotype , Polymerase Chain Reaction , Seizures/metabolism , Seizures/physiopathology , Ubiquitin-Protein Ligases/genetics , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/geneticsABSTRACT
Despite a long tradition of using rats and mice to model epilepsy, several aspects of rodent biology limit their use in large-scale genetic and therapeutic drug screening programs. Neuroscientists interested in vertebrate development and diseases have recently turned to zebrafish (Danio rerio) to overcome these limitations. Zebrafish can be studied at all stages of development and several methods are available for the manipulation of genes in zebrafish. In addition, developing zebrafish larvae can efficiently equilibrate drugs placed in the bathing medium. Taking advantage of these features and adapting electrophysiological recording methods to an agar-immobilized zebrafish preparation, we describe here our efforts to model seizure disorders in zebrafish. We also describe the initial results of a large-scale mutagenesis screen to identify gene mutation(s) that confer seizure resistance. Although the adaptation of zebrafish to epilepsy research is in its early stages, these studies highlight the rapid progress that can be made using this simple vertebrate species.
Subject(s)
Disease Models, Animal , Epilepsy/genetics , Zebrafish/genetics , Amino Acid Sequence , Animals , Humans , Molecular Sequence Data , Mutation/genetics , Seizures/genetics , Survival Analysis , Tenascin/genetics , Zebrafish Proteins/chemistry , Zebrafish Proteins/geneticsABSTRACT
PURPOSE: Zebrafish are a vertebrate organism ideally suited to mutagenesis screening strategies. Although a genetic basis for seizure susceptibility and epilepsy is well established, no efforts have been made to study seizure resistance. Here we describe a novel strategy to isolate seizure-resistant zebrafish mutants from a large-scale mutagenesis screen. METHODS: Seizures were induced with pentylenetetrazole (PTZ). Zebrafish were analyzed between 3 and 7 days postfertilization (dpf). Genome mutations were induced in founders by using N-ethyl-nitrosourea (ENU). Seizure behavior was monitored by using a high-speed camera and quantified by locomotion-tracking software. Electrographic activity was monitored by using a field-recording electrode placed in the optic tectum of agar-immobilized zebrafish. RESULTS: Short-term PTZ exposure elicited a burst-suppression seizure pattern in 3-dpf zebrafish and more complex activity consisting of interictal- and ictal-like discharges at 7 dpf. Prolonged exposure to PTZ induced status epilepticus-like seizure activity and fatality in wild-type zebrafish larvae. With a PTZ survival assay at 6-7 dpf, we identified six zebrafish mutants in a forward-genetic screen covering nearly 2,000 F(2) families. One mutant (s334) also was shown to exhibit reduced behavioral activity on short-term PTZ exposure and an inability to generate long-duration ictal-like discharge. CONCLUSIONS: Zebrafish offers a powerful tool for the identification and study of a genetic basis for seizure resistance.
Subject(s)
Disease Models, Animal , Genetic Testing , Mutagenesis/genetics , Seizures/prevention & control , Zebrafish/genetics , Animals , Behavior, Animal/drug effects , Behavior, Animal/physiology , Genetic Predisposition to Disease/genetics , Larva/genetics , Larva/physiology , Locomotion/drug effects , Locomotion/genetics , Pentylenetetrazole/pharmacology , Seizures/chemically induced , Seizures/epidemiology , Status Epilepticus/chemically induced , Status Epilepticus/prevention & control , Superior Colliculi/physiologyABSTRACT
PURPOSE: In patients with tuberous sclerosis complex (TSC), a wide range of neurologic abnormalities develop, including mental retardation and seizures. Brains from TSC patients are characterized by the presence of cortical tubers, large dysmorphic neurons, and abnormal cytomegalic cells. Although analysis of human TSC brain samples led to the identification of these abnormal cell types, very little is known about how these cells function. In an effort to model TSC-associated CNS abnormalities (and ultimately to analyze the electrophysiologic properties of abnormal cells), we examined Eker rats carrying a Tsc2 mutation. Anatomic studies, including standard histologic stains and immunocytochemistry, were performed on young Eker rats exposed to a carcinogen in utero or aged untreated Eker rats (18-24 months old). METHODS: Pregnant TSC2+/- females were injected once a day with hydroquinone (HQ), and offspring were killed at postnatal day P14 or P28. Coronal tissue sections throughout the CNS were prepared and stained for cresyl violet. In separate studies, brains of old untreated Eker rats were sectioned for anatomic analysis by using standard immunohistochemical techniques. RESULTS: Tissue sections stained with cresyl violet did not reveal any gross differences between HQ-treated Eker (Tsc2Ek/+) rats and siblings (Tsc2+/+). However, two classes of abnormal giant cells were observed in brain sections from untreated aged Eker rats: (a) large dysmorphic pyramid-like cells immunoreactive for NeuN, tuberin, and EAAC-1 in layers IV-VI; and (b) abnormal cytomegalic cells immunoreactive for glial fibrillary acidic protein (GFAP), vimentin, and nestin in deep cortical layers or along the white matter. In addition, large subependymal astrocytomas were observed in four animals. CONCLUSIONS: Our data suggest that cortical tuber formation in Eker rats is a rare event and that prenatal exposure to a nongenotoxic carcinogen such as HQ is not sufficient to induce tuber formation. However, with advanced age, an increased likelihood of astrocytoma formation and the emergence of dysmorphic neurons and cytomegalic cells in the Eker rat brain might exist; each of these abnormalities mimics those seen clinically and could contribute to neurologic problems associated with TSC. Further analysis of this rodent model may be warranted.