Testing of putative antiseizure medications in a preclinical Dravet syndrome zebrafish model.

Dravet syndrome is a severe genetic epilepsy primarily caused by de novo mutations in a voltage-activated sodium channel gene (SCN1A). Patients face life-threatening seizures that are largely resistant to available anti-seizure medications. Preclinical Dravet syndrome animal models are a valuable tool to identify candidate anti-seizure medications for these patients. Among these, scn1lab mutant zebrafish, exhibiting spontaneous seizure-like activity, are particularly amenable to large-scale drug screening. Thus far, we have screened more than 3000 drug candidates in scn1lab zebrafish mutants, identifying valproate, stiripentol, and fenfluramine e.g. Food and Drug Administration-approved drugs, with clinical application in the Dravet syndrome population. Successful phenotypic screening in scn1lab mutant zebrafish is rigorous and consists of two stages: (i) a locomotion-based assay measuring high-velocity convulsive swim behaviour and (ii) an electrophysiology-based assay, using in vivo local field potential recordings, to quantify electrographic seizure-like events. Historically, nearly 90% of drug candidates fail during translation from preclinical models to the clinic. With such a high failure rate, it becomes necessary to address issues of replication and false positive identification. Leveraging our scn1lab zebrafish assays is one approach to address these problems. Here, we curated a list of nine anti-seizure drug candidates recently identified by other groups using preclinical Dravet syndrome models: 1-Ethyl-2-benzimidazolinone, AA43279, chlorzoxazone, donepezil, lisuride, mifepristone, pargyline, soticlestat and vorinostat. First-stage locomotion-based assays in scn1lab mutant zebrafish identified only 1-Ethyl-2-benzimidazolinone, chlorzoxazone and lisuride. However, second-stage local field potential recording assays did not show significant suppression of spontaneous electrographic seizure activity for any of the nine anti-seizure drug candidates. Surprisingly, soticlestat induced frank electrographic seizure-like discharges in wild-type control zebrafish. Taken together, our results failed to replicate clear anti-seizure efficacy for these drug candidates highlighting a necessity for strict scientific standards in preclinical identification of anti-seizure medications.

Host brain environmental influences on transplanted medial ganglionic eminence progenitors.

Interneuron progenitor transplantation can ameliorate disease symptoms in a variety of neurological disorders. The strategy is based on transplantation of embryonic medial ganglionic eminence (MGE) progenitors. Elucidating how host brain environment influences the integration of interneuron progenitors is critical for optimizing this strategy across different disease states. Here, we systematically evaluated the influence of age and brain region on survival, migration, and differentiation of transplant-derived cells. We find that early postnatal MGE transplantation yields superior survival and more extensive migratory capabilities compared to transplantation during the juvenile or adult stages. MGE progenitors migrate more widely in the cortex compared to the hippocampus. Maturation to interneuron subtypes is regulated by age and brain region. MGE progenitors transplanted into the dentate gyrus sub-region of the early postnatal hippocampus can differentiate into astrocytes. Our results suggest that the host brain environment critically regulates survival, spatial distribution, and maturation of MGE-derived interneurons following transplantation. These findings inform and enable optimal conditions for interneuron transplant therapies.

Cell therapy for neurological disorders: Progress towards an embryonic medial ganglionic eminence progenitor-based treatment.

Impairment of development, migration, or function of inhibitory interneurons are key features of numerous circuit-based neurological disorders, such as epilepsy. From a therapeutic perspective, symptomatic treatment of these disorders often relies upon drugs or deep brain stimulation approaches to provide a general enhancement of GABA-mediated inhibition. A more effective strategy to target these pathological circuits and potentially provide true disease-modifying therapy, would be to selectively add new inhibitory interneurons into these circuits. One such strategy, using embryonic medial ganglionic (MGE) progenitor cells as a source of a unique sub-population of interneurons, has already proven effective as a cell transplantation therapy in a variety of preclinical models of neurological disorders, especially in mouse models of acquired epilepsy. Here we will discuss the evolution of this interneuron-based transplantation therapy in acquired epilepsy models, with an emphasis on the recent adaptation of MGE progenitor cells for xenotransplantation into larger mammals.

Xenotransplantation of porcine progenitor cells in an epileptic California sea lion (Zalophus californianus): illustrative case.

BACKGROUND: Domoic acid (DA) is a naturally occurring neurotoxin harmful to marine animals and humans. California sea lions exposed to DA in prey during algal blooms along the Pacific coast exhibit significant neurological symptoms, including epilepsy with hippocampal atrophy. OBSERVATIONS: Here the authors describe a xenotransplantation procedure to deliver interneuron progenitor cells into the damaged hippocampus of an epileptic sea lion with suspected DA toxicosis. The sea lion has had no evidence of seizures after the procedure, and clinical measures of well-being, including weight and feeding habits, have stabilized. LESSONS: These preliminary results suggest xenotransplantation has improved the quality of life for this animal and holds tremendous therapeutic promise.

Clemizole and trazodone are effective antiseizure treatments in a zebrafish model of STXBP1 disorder.

CRISPR-Cas9-generated zebrafish carrying a 12 base-pair deletion in stxbpb1b, a paralog sharing 79% amino acid sequence identity with human, exhibit spontaneous electrographic seizures during larval stages of development. Zebrafish stxbp1b mutants provide an efficient preclinical platform to test antiseizure therapeutics. The present study was designed to test antiseizure medications approved for clinical use and two recently identified repurposed drugs with antiseizure activity. Larval homozygous stxbp1b zebrafish (4 days postfertilization (dpf)) were agarose-embedded and monitored for electrographic seizure activity using a local field recording electrode placed in midbrain. Frequency of ictal-like events was evaluated at baseline and following 45 min of continuous drug exposure (1 mM, bath application). Analysis was performed on coded files by an experimenter blinded to drug treatment and genotype. Phenytoin (PHT), valproate (VPA), ethosuximide (ESX), levetiracetam (LEV), and diazepam (DZP) had no effect on the ictal-like event frequency in stxbp1b mutant zebrafish. Clemizole and trazodone decreased ictal-like event frequency in stxbp1b mutant zebrafish by 80% and 83%, respectively. These results suggest that repurposed drugs with serotonin receptor-binding affinities could be effective antiseizure treatments. Clemizole and trazodone were previously identified in a larval zebrafish model for Dravet syndrome. Based primarily on these preclinical zebrafish studies, compassionate-use and double-blind clinical trials with both drugs have progressed. The present study extends this approach to a preclinical zebrafish model representing STXBP1 (syntaxin-binding protein 1)-related disorders and suggests that future clinical studies may be warranted.

Hippocampal gamma and sharp-wave ripple oscillations are altered in a Cntnap2 mouse model of autism spectrum disorder.

Impaired synaptic neurotransmission may underly circuit alterations contributing to behavioral autism spectrum disorder (ASD) phenotypes. A critical component of impairments reported in somatosensory and prefrontal cortex of ASD mouse models are parvalbumin (PV)-expressing fast-spiking interneurons. However, it remains unknown whether PV interneurons mediating hippocampal networks crucial to navigation and memory processing are similarly impaired. Using PV-labeled transgenic mice, a battery of behavioral assays, in vitro patch-clamp electrophysiology, and in vivo 32-channel silicon probe local field potential recordings, we address this question in a Cntnap2-null mutant mouse model representing a human ASD risk factor gene. Cntnap2-/- mice show a reduction in hippocampal PV interneuron density, reduced inhibitory input to CA1 pyramidal cells, deficits in spatial discrimination ability, and frequency-dependent circuit changes within the hippocampus, including alterations in gamma oscillations, sharp-wave ripples, and theta-gamma modulation. Our findings highlight hippocampal involvement in ASD and implicate interneurons as a potential therapeutical target.

Maximally selective single-cell target for circuit control in epilepsy models.

Neurological and psychiatric disorders are associated with pathological neural dynamics. The fundamental connectivity patterns of cell-cell communication networks that enable pathological dynamics to emerge remain unknown. Here, we studied epileptic circuits using a newly developed computational pipeline that leveraged single-cell calcium imaging of larval zebrafish and chronically epileptic mice, biologically constrained effective connectivity modeling, and higher-order motif-focused network analysis. We uncovered a novel functional cell type that preferentially emerged in the preseizure state, the superhub, that was unusually richly connected to the rest of the network through feedforward motifs, critically enhancing downstream excitation. Perturbation simulations indicated that disconnecting superhubs was significantly more effective in stabilizing epileptic circuits than disconnecting hub cells that were defined traditionally by connection count. In the dentate gyrus of chronically epileptic mice, superhubs were predominately modeled adult-born granule cells. Collectively, these results predict a new maximally selective and minimally invasive cellular target for seizure control.

A zebrafish-centric approach to antiepileptic drug development.

Danio rerio (zebrafish) are a powerful experimental model for genetic and developmental studies. Adaptation of zebrafish to study seizures was initially established using the common convulsant agent pentylenetetrazole (PTZ). Larval PTZ-exposed zebrafish exhibit clear behavioral convulsions and abnormal electrographic activity, reminiscent of interictal and ictal epileptiform discharge. By using this model, our laboratory developed simple locomotion-based and electrophysiological assays to monitor and quantify seizures in larval zebrafish. Zebrafish also offer multiple advantages for rapid genetic manipulation and high-throughput phenotype-based drug screening. Combining these seizure assays with genetically modified zebrafish that represent Dravet syndrome, a rare genetic epilepsy, ultimately contributed to a phenotype-based screen of over 3500 drugs. Several drugs identified in these zebrafish screens are currently in clinical or compassionate-use trials. The emergence of this 'aquarium-to-bedside' approach suggests that broader efforts to adapt and improve upon this zebrafish-centric strategy can drive a variety of exciting new discoveries.

In vivo calcium imaging reveals disordered interictal network dynamics in epileptic stxbp1b zebrafish.

STXBP1 mutations are associated with encephalopathy, developmental delay, intellectual disability, and epilepsy. While neural networks are known to operate at a critical state in the healthy brain, network behavior during pathological epileptic states remains unclear. Examining activity during periods between well-characterized ictal-like events (i.e., interictal period) could provide a valuable step toward understanding epileptic networks. To study these networks in the context of STXBP1 mutations, we combine a larval zebrafish model with in vivo fast confocal calcium imaging and extracellular local field potential recordings. Stxbp1b mutants display transient periods of elevated activity among local clusters of interacting neurons. These network "cascade" events were significantly larger in size and duration in mutants. At mesoscale resolution, cascades exhibit neurodevelopmental abnormalities. At single-cell scale, we describe spontaneous hyper-synchronized neuronal ensembles. That calcium imaging reveals uniquely disordered brain states during periods between pathological ictal-like seizure events is striking and represents a potential interictal biomarker.

Phenotypic analysis of catastrophic childhood epilepsy genes.

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 ∼3300 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.

Enhancing glucose metabolism via gluconeogenesis is therapeutic in a zebrafish model of Dravet syndrome.

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.

Interneuron Origins in the Embryonic Porcine Medial Ganglionic Eminence.

Interneurons contribute to the complexity of neural circuits and maintenance of normal brain function. Rodent interneurons originate in embryonic ganglionic eminences, but developmental origins in other species are less understood. Here, we show that transcription factor expression patterns in porcine embryonic subpallium are similar to rodents, delineating a distinct medial ganglionic eminence (MGE) progenitor domain. On the basis of Nkx2.1, Lhx6, and Dlx2 expression, in vitro differentiation into neurons expressing GABA, and robust migratory capacity in explant assays, we propose that cortical and hippocampal interneurons originate from a porcine MGE region. Following xenotransplantation into adult male and female rat hippocampus, we further demonstrate that porcine MGE progenitors, like those from rodents, migrate and differentiate into morphologically distinct interneurons expressing GABA. Our findings reveal that basic rules for interneuron development are conserved across species, and that porcine embryonic MGE progenitors could serve as a valuable source for interneuron-based xenotransplantation therapies.SIGNIFICANCE STATEMENT Here we demonstrate that porcine medial ganglionic eminence, like rodents, exhibit a distinct transcriptional and interneuron-specific antibody profile, in vitro migratory capacity and are amenable to xenotransplantation. This is the first comprehensive examination of embryonic interneuron origins in the pig; and because a rich neurodevelopmental literature on embryonic mouse medial ganglionic eminence exists (with some additional characterizations in other species, e.g., monkey and human), our work allows direct neurodevelopmental comparisons with this literature.

Phenotype-Based Screening of Synthetic Cannabinoids in a Dravet Syndrome Zebrafish Model.

Dravet syndrome is a catastrophic epilepsy of childhood, characterized by cognitive impairment, severe seizures, and increased risk for sudden unexplained death in epilepsy (SUDEP). Although refractory to conventional antiepileptic drugs, emerging preclinical and clinical evidence suggests that modulation of the endocannabinoid system could be therapeutic in these patients. Preclinical research on this topic is limited as cannabis, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), are designated by United States Drug Enforcement Agency (DEA) as illegal substances. In this study, we used a validated zebrafish model of Dravet syndrome, scn1lab homozygous mutants, to screen for anti-seizure activity in a commercially available library containing 370 synthetic cannabinoid (SC) compounds. SCs are intended for experimental use and not restricted by DEA designations. Primary phenotype-based screening was performed using a locomotion-based assay in 96-well plates, and a secondary local field potential recording assay was then used to confirm suppression of electrographic epileptiform events. Identified SCs with anti-seizure activity, in both assays, included five SCs structurally classified as indole-based cannabinoids JWH 018 N-(5-chloropentyl) analog, JWH 018 N-(2-methylbutyl) isomer, 5-fluoro PB-22 5-hydroxyisoquinoline isomer, 5-fluoro ADBICA, and AB-FUBINACA 3-fluorobenzyl isomer. Our approach demonstrates that two-stage phenotype-based screening in a zebrafish model of Dravet syndrome successfully identifies SCs with anti-seizure activity.

Zebrafish studies identify serotonin receptors mediating antiepileptic activity in Dravet syndrome.

Dravet syndrome is a life-threatening early-onset epilepsy not well controlled by antiepileptic drugs. Drugs that modulate serotonin (5-HT) signalling, including clemizole, locaserin, trazodone and fenfluramine, have recently emerged as potential treatment options for Dravet syndrome. To investigate the serotonin receptors that could moderate this antiepileptic activity, we designed and synthesized 28 novel analogues of clemizole, obtained receptor binding affinity profiles, and performed in vivo screening in a scn1lab mutant zebrafish (Danio rerio) model which recapitulates critical clinical features of Dravet syndrome. We discovered three clemizole analogues with 5-HT receptor binding that exert powerful antiepileptic activity. Based on structure-activity relationships and medicinal chemistry-based analysis, we then screened an additional set of known 5-HT receptor specific drug candidates. Integrating our in vitro and in vivo data implicates 5-HT2B receptors as a critical mediator in the mechanism of seizure suppression observed in Dravet syndrome patients treated with 5-HT modulating drugs.

Network Properties Revealed during Multi-Scale Calcium Imaging of Seizure Activity in Zebrafish.

Seizures are characterized by hypersynchronization of neuronal networks. Understanding these networks could provide a critical window for therapeutic control of recurrent seizure activity, i.e., epilepsy. However, imaging seizure networks has largely been limited to microcircuits in vitro or small "windows" in vivo. Here, we combine fast confocal imaging of genetically encoded calcium indicator (GCaMP)-expressing larval zebrafish with local field potential (LFP) recordings to study epileptiform events at whole-brain and single-neuron levels in vivo. Using an acute seizure model (pentylenetetrazole, PTZ), we reliably observed recurrent electrographic ictal-like events associated with generalized activation of all major brain regions and uncovered a well-preserved anterior-to-posterior seizure propagation pattern. We also examined brain-wide network synchronization and spatiotemporal patterns of neuronal activity in the optic tectum microcircuit. Brain-wide and single-neuronal level analysis of PTZ-exposed and 4-aminopyridine (4-AP)-exposed zebrafish revealed distinct network dynamics associated with seizure and non-seizure hyperexcitable states, respectively. Neuronal ensembles, comprised of coactive neurons, were also uncovered during interictal-like periods. Taken together, these results demonstrate that macro- and micro-network calcium motifs in zebrafish may provide a greater understanding of epilepsy.

Preclinical Animal Models for Dravet Syndrome: Seizure Phenotypes, Comorbidities and Drug Screening.

Epilepsy is a common chronic neurological disease affecting almost 3 million people in the United States and 50 million people worldwide. Despite availability of more than two dozen FDA-approved anti-epileptic drugs (AEDs), one-third of patients fail to receive adequate seizure control. Specifically, pediatric genetic epilepsies are often the most severe, debilitating and pharmaco-resistant forms of epilepsy. Epileptic syndromes share a common symptom of unprovoked seizures. While some epilepsies/forms of epilepsy are the result of acquired insults such as head trauma, febrile seizure, or viral infection, others have a genetic basis. The discovery of epilepsy associated genes suggests varied underlying pathologies and opens the door for development of new "personalized" treatment options for each genetic epilepsy. Among these, Dravet syndrome (DS) has received substantial attention for both the pre-clinical and early clinical development of novel therapeutics. Despite these advances, there is no FDA-approved treatment for DS. Over 80% of patients diagnosed with DS carry a de novo mutation within the voltage-gated sodium channel gene SCN1A and these patients suffer with drug resistant and life-threatening seizures. Here we will review the preclinical animal models for DS featuring inactivation of SCN1A (including zebrafish and mice) with an emphasis on seizure phenotypes and behavioral comorbidities. Because many drugs fail somewhere between initial preclinical discovery and clinical trials, it is equally important that we understand how these models respond to known AEDs. As such, we will also review the available literature and recent drug screening efforts using these models with a focus on assay protocols and predictive pharmacological profiles. Validation of these preclinical models is a critical step in our efforts to efficiently discover new therapies for these patients. The behavioral and electrophysiological drug screening assays in zebrafish will be discussed in detail including specific examples from our laboratory using a zebrafish scn1 mutant and a summary of the nearly 3000 drugs screened to date. As the discovery and development phase rapidly moves from the lab-to-the-clinic for DS, it is hoped that this preclinical strategy offers a platform for how to approach any genetic epilepsy.

Publisher Correction: PAFAH1B1 haploinsufficiency disrupts GABA neurons and synaptic E/I balance in the dentate gyrus.

A correction to this article has been published and is linked from the HTML version of this paper. The error has not been fixed in the paper.

Dlx1 and Dlx2 Promote Interneuron GABA Synthesis, Synaptogenesis, and Dendritogenesis.

The postnatal functions of the Dlx1&2 transcription factors in cortical interneurons (CINs) are unknown. Here, using conditional Dlx1, Dlx2, and Dlx1&2 knockouts (CKOs), we defined their roles in specific CINs. The CKOs had dendritic, synaptic, and survival defects, affecting even PV+ CINs. We provide evidence that DLX2 directly drives Gad1, Gad2, and Vgat expression, and show that mutants had reduced mIPSC amplitude. In addition, the mutants formed fewer GABAergic synapses on excitatory neurons and had reduced mIPSC frequency. Furthermore, Dlx1/2 CKO had hypoplastic dendrites, fewer excitatory synapses, and reduced excitatory input. We provide evidence that some of these phenotypes were due to reduced expression of GRIN2B (a subunit of the NMDA receptor), a high confidence Autism gene. Thus, Dlx1&2 coordinate key components of CIN postnatal development by promoting their excitability, inhibitory output, and survival.

Mutations of conserved non-coding elements of PITX2 in patients with ocular dysgenesis and developmental glaucoma.

Mutations in FOXC1 and PITX2 constitute the most common causes of ocular anterior segment dysgenesis (ASD), and confer a high risk for secondary glaucoma. The genetic causes underlying ASD in approximately half of patients remain unknown, despite many of them being screened by whole exome sequencing. Here, we performed whole genome sequencing on DNA from two affected individuals from a family with dominantly inherited ASD and glaucoma to identify a 748-kb deletion in a gene desert that contains conserved putative PITX2 regulatory elements. We used CRISPR/Cas9 to delete the orthologous region in zebrafish in order to test the pathogenicity of this structural variant. Deletion in zebrafish reduced pitx2 expression during development and resulted in shallow anterior chambers. We screened additional patients for copy number variation of the putative regulatory elements and found an overlapping deletion in a second family and in a potentially-ancestrally-related index patient with ASD and glaucoma. These data suggest that mutations affecting conserved non-coding elements of PITX2 may constitute an important class of mutations in patients with ASD for whom the molecular cause of their disease have not yet been identified. Improved functional annotation of the human genome and transition to sequencing of patient genomes instead of exomes will be required before the magnitude of this class of mutations is fully understood.