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.

[1]  H. Ikeda,et al.  Loss-of-function mutations of STXBP1 in patients with epileptic encephalopathy , 2016, Brain and Development.

[2]  Brian P. Grone,et al.  Divergent evolution of two corticotropin-releasing hormone (CRH) genes in teleost fishes , 2015, Front. Neurosci..

[3]  H. Sive,et al.  Challenges in understanding psychiatric disorders and developing therapeutics: a role for zebrafish , 2015, Disease Models & Mechanisms.

[4]  Brian P. Grone,et al.  A second corticotropin‐releasing hormone gene (CRH2) is conserved across vertebrate classes and expressed in the hindbrain of a basal Neopterygian fish, the spotted gar (Lepisosteus oculatus) , 2015, The Journal of comparative neurology.

[5]  Marilisa G. Elrod,et al.  Sleep Differences Among Children With Autism Spectrum Disorders and Typically Developing Peers: A Meta-analysis , 2015, Journal of developmental and behavioral pediatrics : JDBP.

[6]  A. Schier,et al.  The study of psychiatric disease genes and drugs in zebrafish , 2015, Current Opinion in Neurobiology.

[7]  C. Betsholtz,et al.  Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish. , 2015, Developmental cell.

[8]  Uwe Irion,et al.  Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system , 2014, Development.

[9]  A. Basu,et al.  A novel de novo STXBP1 mutation is associated with mitochondrial complex I deficiency and late-onset juvenile-onset parkinsonism , 2014, neurogenetics.

[10]  J. Roach,et al.  Mutations in STX1B, encoding a presynaptic protein, cause fever-associated epilepsy syndromes , 2014, Nature Genetics.

[11]  P. Drapeau,et al.  Fishing for causes and cures of motor neuron disorders , 2014, Disease Models & Mechanisms.

[12]  J. Shendure,et al.  GABRA1 and STXBP1: Novel genetic causes of Dravet syndrome , 2014, Neurology.

[13]  C. Walsh,et al.  Mutations in QARS, encoding glutaminyl-tRNA synthetase, cause progressive microcephaly, cerebral-cerebellar atrophy, and intractable seizures. , 2014, American journal of human genetics.

[14]  E. van Wijk,et al.  CNNM2 Mutations Cause Impaired Brain Development and Seizures in Patients with Hypomagnesemia , 2014, PLoS genetics.

[15]  O. Stiedl,et al.  Munc18-1 haploinsufficiency results in enhanced anxiety-like behavior as determined by heart rate responses in mice , 2014, Behavioural Brain Research.

[16]  Haley A. Coleman,et al.  Neuropeptidergic Signaling Partitions Arousal Behaviors in Zebrafish , 2014, The Journal of Neuroscience.

[17]  Stephen W. Wilson,et al.  Left-Right Asymmetry Is Required for the Habenulae to Respond to Both Visual and Olfactory Stimuli , 2014, Current Biology.

[18]  V. Wong,et al.  Folinic acid responsive epilepsy in Ohtahara syndrome caused by STXBP1 mutation. , 2014, Pediatric neurology.

[19]  A. Munnich,et al.  A novel mutation in STXBP1 causing epileptic encephalopathy (late onset infantile spasms) with partial respiratory chain complex IV deficiency. , 2013, European journal of medical genetics.

[20]  T. Südhof,et al.  Neurotransmitter Release: The Last Millisecond in the Life of a Synaptic Vesicle , 2013, Neuron.

[21]  Scott C. Baraban,et al.  Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet Syndrome treatment , 2013, Nature Communications.

[22]  Jeffry D. Sander,et al.  Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System , 2013, PloS one.

[23]  P. Beales,et al.  Generation and validation of a zebrafish model of EAST (epilepsy, ataxia, sensorineural deafness and tubulopathy) syndrome , 2013, Disease Models & Mechanisms.

[24]  S. Baraban.  Forebrain electrophysiological recording in larval zebrafish. , 2013, Journal of visualized experiments : JoVE.

[25]  Jeffry D. Sander,et al.  Efficient In Vivo Genome Editing Using RNA-Guided Nucleases , 2013, Nature Biotechnology.

[26]  L. Appelbaum,et al.  Circadian clocks, rhythmic synaptic plasticity and the sleep-wake cycle in zebrafish , 2012, Front. Neural Circuits.

[27]  S. Huffel,et al.  Cardiac autonomic dysfunction in West syndrome , 2012, Epilepsy Research.

[28]  A. Gillespie,et al.  A novel zebrafish model of hyperthermia-induced seizures reveals a role for TRPV4 channels and NMDA-type glutamate receptors , 2012, Experimental Neurology.

[29]  N. Divecha,et al.  Impaired neural development in a zebrafish model for Lowe syndrome , 2011, Human molecular genetics.

[30]  A. Kaminska,et al.  Epileptic and nonepileptic features in patients with early onset epileptic encephalopathy and STXBP1 mutations , 2011, Epilepsia.

[31]  M. Granato,et al.  Chemical modulation of memory formation in larval zebrafish , 2011, Proceedings of the National Academy of Sciences.

[32]  A. Amores,et al.  Genome Evolution and Meiotic Maps by Massively Parallel DNA Sequencing: Spotted Gar, an Outgroup for the Teleost Genome Duplication , 2011, Genetics.

[33]  J. Lacaille,et al.  Intellectual disability without epilepsy associated with STXBP1 disruption , 2011, European Journal of Human Genetics.

[34]  M. Kerr,et al.  Evolution and management of Lennox-Gastaut syndrome through adolescence and into adulthood: are seizures always the primary issue? , 2011, Epileptic disorders : international epilepsy journal with videotape.

[35]  H. Oguni,et al.  STXBP1 mutations cause not only Ohtahara syndrome but also West syndrome—Result of Japanese cohort study , 2010, Epilepsia.

[36]  Xiayang Xie,et al.  Knockdown of zebrafish Lgi1a results in abnormal development, brain defects and a seizure-like behavioral phenotype. , 2010, Human molecular genetics.

[37]  M. Westerfield,et al.  Inaugural Article: Pineal-specific agouti protein regulates teleost background adaptation , 2010 .

[38]  I. Scheffer,et al.  Clinical spectrum of early-onset epileptic encephalopathies associated with STXBP1 mutations , 2010, Neurology.

[39]  Aristides B. Arrenberg,et al.  Optogenetic Localization and Genetic Perturbation of Saccade-Generating Neurons in Zebrafish , 2010, The Journal of Neuroscience.

[40]  P. Page-McCaw,et al.  Cardiac response to startle stimuli in larval zebrafish: sympathetic and parasympathetic components. , 2010, American journal of physiology. Regulatory, integrative and comparative physiology.

[41]  Drena Dobbs,et al.  ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool , 2010, Nucleic Acids Res..

[42]  D. Parichy,et al.  Normal table of postembryonic zebrafish development: Staging by externally visible anatomy of the living fish , 2009, Developmental dynamics : an official publication of the American Association of Anatomists.

[43]  A. Perkins,et al.  Evolution of gene function and regulatory control after whole-genome duplication: comparative analyses in vertebrates. , 2009, Genome research.

[44]  M. Scott,et al.  A homozygous mutation in human PRICKLE1 causes an autosomal-recessive progressive myoclonus epilepsy-ataxia syndrome. , 2008, American journal of human genetics.

[45]  Naomichi Matsumoto,et al.  De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy , 2008, Nature Genetics.

[46]  H. Burgess,et al.  Modulation of locomotor activity in larval zebrafish during light adaptation , 2007, Journal of Experimental Biology.

[47]  S. Baraban,et al.  A Large‐scale Mutagenesis Screen to Identify Seizure‐resistant Zebrafish , 2007, Epilepsia.

[48]  T. Südhof,et al.  Munc18-1 binds directly to the neuronal SNARE complex , 2007, Proceedings of the National Academy of Sciences.

[49]  O. Jaillon,et al.  Gene loss and evolutionary rates following whole-genome duplication in teleost fishes. , 2006, Molecular biology and evolution.

[50]  Shunsuke Ohtahara,et al.  Ohtahara syndrome: With special reference to its developmental aspects for differentiating from early myoclonic encephalopathy , 2006, Epilepsy Research.

[51]  Melissa A. Wright,et al.  Embryonic and larval expression of zebrafish voltage‐gated sodium channel α‐subunit genes , 2006 .

[52]  I. Zhdanova,et al.  Sleep in zebrafish. , 2006, Zebrafish.

[53]  I. Jackson,et al.  Regulation of pigmentation in zebrafish melanophores. , 2006, Pigment cell research.

[54]  Thorsten Schwerte,et al.  Development of the sympatho-vagal balance in the cardiovascular system in zebrafish (Danio rerio) characterized by power spectrum and classical signal analysis , 2006, Journal of Experimental Biology.

[55]  Michael R. Taylor,et al.  Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression , 2005, Neuroscience.

[56]  Charles E. Chapple,et al.  Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype , 2004, Nature.

[57]  A. Meyer,et al.  Phylogenetic Timing of the Fish-Specific Genome Duplication Correlates with the Diversification of Teleost Fish , 2004, Journal of Molecular Evolution.

[58]  Alan Christoffels,et al.  Fugu genome analysis provides evidence for a whole-genome duplication early during the evolution of ray-finned fishes. , 2004, Molecular biology and evolution.

[59]  Erik M Jorgensen,et al.  Defects in synaptic vesicle docking in unc-18 mutants , 2003, Nature Neuroscience.

[60]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[61]  Thomas C. Südhof,et al.  Munc18-1 Promotes Large Dense-Core Vesicle Docking , 2001, Neuron.

[62]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[63]  T. Südhof,et al.  Synaptic assembly of the brain in the absence of neurotransmitter secretion. , 2000, Science.

[64]  William A. Harris,et al.  Genetic Disorders of Vision Revealed by a Behavioral Screen of 400 Essential Loci in Zebrafish , 1999, The Journal of Neuroscience.

[65]  A. Force,et al.  Preservation of duplicate genes by complementary, degenerative mutations. , 1999, Genetics.

[66]  B. Pelster,et al.  Disruption of hemoglobin oxygen transport does not impact oxygen-dependent physiological processes in developing embryos of zebra fish (Danio rerio). , 1996, Circulation research.

[67]  J B Hurley,et al.  A behavioral screen for isolating zebrafish mutants with visual system defects. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[68]  G. Rubin,et al.  Mutations in the drosophila Rop gene suggest a function in general secretion and synaptic transmission , 1994, Neuron.

[69]  A. Hughes.  The evolution of functionally novel proteins after gene duplication , 1994, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[70]  P. De Camilli,et al.  A rat brain Sec1 homologue related to Rop and UNC18 interacts with syntaxin. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[71]  R. Scheller,et al.  n-Sec1: a neural-specific syntaxin-binding protein. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[72]  T. Südhof,et al.  Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin , 1993, Nature.

[73]  J. Eisen,et al.  Development of the neural crest in the zebrafish. , 1993, Developmental biology.

[74]  L. Leinonen,et al.  Melatonin, cortisol and body temperature rhythms in Lennox-Gastaut patients with or without circadian rhythm sleep disorders , 1993, Journal of Neurology.

[75]  J. Barker,et al.  Pentylenetetrazol and penicillin are selective antagonists of GABA-mediated post-synaptic inhibition in cultured mammalian neurones , 1977, Nature.

[76]  J. Ball,et al.  Evidence for a dual pituitary control of teleost melanophores. , 1975, General and comparative endocrinology.

[77]  L. Hogben,et al.  The Pigmentary Effector System. VI. The Dual Character of Endocrine Co-Ordination in Amphibian Colour Change , 1931 .

[78]  De novo mutations in epileptic encephalopathies , 2013 .

[79]  Melissa A. Wright,et al.  Embryonic and larval expression of zebrafish voltage-gated sodium channel alpha-subunit genes. , 2006, Developmental dynamics : an official publication of the American Association of Anatomists.

[80]  A. Force,et al.  The probability of duplicate gene preservation by subfunctionalization. , 2000, Genetics.

[81]  M. Westerfield.  The zebrafish book : a guide for the laboratory use of zebrafish (Danio rerio) , 1995 .

[82]  HighWire Press.  The journal of neuroscience : the official journal of the Society for Neuroscience. , 1981 .