Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1) Mutant Zebrafish.

Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1), are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b) have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing "dark-flash" visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations

Neuronal birthdate reveals topography in a vestibular brainstem circuit for gaze stabilization

Across the nervous system, neurons with similar attributes are topographically organized. This topography reflects developmental pressures. Oddly, vestibular (balance) nuclei are thought to be disorganized. By measuring activity in birthdated neurons, we revealed a functional map within the central vestibular projection nucleus that stabilizes gaze in the larval zebrafish. We first discovered that both somatic position and stimulus selectivity follow projection neuron birthdate. Next, with electron microscopy and loss-of-function assays, we found that patterns of peripheral innervation to projection neurons were similarly organized by birthdate. Finally, birthdate revealed spatial patterns of axonal arborization and synapse formation to projection neuron outputs. Collectively, we find that development reveals previously hidden organization to the input, processing, and output layers of a highly conserved vertebrate sensorimotor circuit. The spatial and temporal attributes we uncover constrain the developmental mechanisms that may specify the fate, function, and organization of vestibulo-ocular reflex neurons. More broadly, our data suggest that, like invertebrates, temporal mechanisms may assemble vertebrate sensorimotor architecture

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

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

Clemizole and modulators of serotonin signalling suppress seizures in Dravet syndrome.

Dravet syndrome is a catastrophic childhood epilepsy with early-onset seizures, delayed language and motor development, sleep disturbances, anxiety-like behaviour, severe cognitive deficit and an increased risk of fatality. It is primarily caused by de novo mutations of the SCN1A gene encoding a neuronal voltage-activated sodium channel. Zebrafish with a mutation in the SCN1A homologue recapitulate spontaneous seizure activity and mimic the convulsive behavioural movements observed in Dravet syndrome. Here, we show that phenotypic screening of drug libraries in zebrafish scn1 mutants rapidly and successfully identifies new therapeutics. We demonstrate that clemizole binds to serotonin receptors and its antiepileptic activity can be mimicked by drugs acting on serotonin signalling pathways e.g. trazodone and lorcaserin. Coincident with these zebrafish findings, we treated five medically intractable Dravet syndrome patients with a clinically-approved serotonin receptor agonist (lorcaserin, Belviq®) and observed some promising results in terms of reductions in seizure frequency and/or severity. Our findings demonstrate a rapid path from preclinical discovery in zebrafish, through target identification, to potential clinical treatments for Dravet syndrome

SAMPL is a high-throughput solution to study unconstrained vertical behavior in small animals

Summary: Balance and movement are impaired in many neurological disorders. Recent advances in behavioral monitoring provide unprecedented access to posture and locomotor kinematics but without the throughput and scalability necessary to screen candidate genes/potential therapeutics. Here, we present a scalable apparatus to measure posture and locomotion (SAMPL). SAMPL includes extensible hardware and open-source software with real-time processing and can acquire data from D. melanogaster, C. elegans, and D. rerio as they move vertically. Using SAMPL, we define how zebrafish balance as they navigate vertically and discover small but systematic variations among kinematic parameters between genetic backgrounds. We demonstrate SAMPL’s ability to resolve differences in posture and navigation as a function of effect size and data gathered, providing key data for screens. SAMPL is therefore both a tool to model balance and locomotor disorders and an exemplar of how to scale apparatus to support screens

Zebrafish <i>stxbp1a</i> CRISPR/Cas9 mutant allele.

<p>(A) Sites of human and zebrafish mutations in highly conserved Stxbp1 sequence. Human STXBP1A protein sequence was aligned to zebrafish Stxbp1a and Stxbp1b. Black background indicates amino acid residues that are similar in all three proteins (BLOSUM 62). Grey background indicates amino acid residues that are similar between two of the three proteins. The salmon-colored background indicates the position of the deletion mutation in zebrafish mutant alleles. (B) Alignment of the mutant <i>stxbp1a</i>^s3000 allele sequence (top) with wild-type zebrafish <i>stxbp1a</i> sequence (bottom). The CRISPR/Cas9 target site is shown as a wide purple arrow below the plot. The site of the 4bp deletion <i>stxbp1a</i>^s3000 allele is highlighted in salmon, and corresponds to amino acids 211–212 highlighted in (A). (C) Alignment of the mutant <i>stxbp1b</i>^<i>s3001</i> allele sequence with wild-type zebrafish <i>stxbp1b</i> sequence.</p

Ontogeny of <i>stxbp1a</i> and <i>stxbp1b</i> expression in zebrafish larvae.

<p>Wild-type zebrafish were probed for expression of <i>stxbp1a</i> mRNA (A-E) or <i>stxbp1b</i> mRNA (F-J). Abbreviations: CeP: cerebellar plate, Ha: habenula, IR: inner retina, MO: medulla oblongata, OB: olfactory bulb, P: pallium, Ret: retina, SC: spinal cord, Tel: telencephalon, TeO: optic tectum, Scale bar = 100μm.</p

Morphology of <i>stxbp1a</i>^s3000 mutant zebrafish.

<p>(A) Heterozygous <i>stxbp1a</i>^{<i>s3000/+</i>} mutant larvae (5 dpf) are morphologically indistinguishable from wild-type siblings. Homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae are immobile and fail to hatch out of the chorion. Scale bar = 500 μm. (B) Homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae (n = 10) removed from their chorions are not significantly different in length from their siblings (n = 30; p = 0.0592, two-tailed t-test). (C-D) At 5 dpf, the dorsal surface of homozygous <i>stxbp1a</i>^{<i>s3000/s3000</i>} mutant larvae that were removed from their chorions at 2 dpf (D) show dispersed melanin and foreshortened craniofacial structure compared to siblings (C). Scale bar = 300 μm.</p