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Deficiency of the ywhaz gene, involved in neurodevelopmental disorders, alters brain activity and behaviour in zebrafish

Abstract

Genetic variants in YWHAZ contribute to psychiatric disorders such as autism spectrum disorder and schizophrenia, and have been related to an impaired neurodevelopment in humans and mice. Here, we have used zebrafish to investigate the mechanisms by which YWHAZ contributes to neurodevelopmental disorders. We observed that ywhaz expression was pan-neuronal during developmental stages and restricted to Purkinje cells in the adult cerebellum, cells that are described to be reduced in number and size in autistic patients. We then performed whole-brain imaging in wild-type and ywhaz CRISPR/Cas9 knockout (KO) larvae and found altered neuronal activity and connectivity in the hindbrain. Adult ywhaz KO fish display decreased levels of monoamines in the hindbrain and freeze when exposed to novel stimuli, a phenotype that can be reversed with drugs that target monoamine neurotransmission. These findings suggest an important role for ywhaz in establishing neuronal connectivity during development and modulating both neurotransmission and behaviour in adults.

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Fig. 1: Whole-brain imaging methods.
Fig. 2: ywhaz mRNA is widely expressed during zebrafish development but restricted to the Purkinje cells in the cerebellum in adults.
Fig. 3: Decreased coherence in neuronal activity and decreased connectivity in the hindbrain of zebrafish ywhaz−/− larvae.
Fig. 4: Alterations in the monoamine neurotransmission in the hindbrain of adult KO.
Fig. 5: Treatment with fluoxetine and quinpirole reverses the freezing behaviour observed in ywhaz−/− mutants.

References

  1. Jia Y, Yu X, Zhang B, Yuan Y, Xu Q, Shen Y, et al. An association study between polymorphisms in three genes of 14-3-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein) family and paranoid schizophrenia in northern Chinese population. Eur Psychiatry. 2004;19:377–9.

    PubMed  Article  Google Scholar 

  2. Torrico B, Antón-Galindo E, Fernàndez-Castillo N, Rojo-Francàs E, Ghorbani S, Pineda-Cirera L, et al. Involvement of the 14-3-3 gene family in autism spectrum disorder and schizophrenia: genetics, transcriptomics and functional analyses. J Clin Med. 2020;9:1851.

    CAS  PubMed Central  Article  Google Scholar 

  3. Toma C, Torrico B, Hervás A, Valdés-Mas R, Tristán-Noguero A, Padillo V, et al. Exome sequencing in multiplex autism families suggests a major role for heterozygous truncating mutations. Mol Psychiatry. 2014;19:784–90.

    CAS  PubMed  Article  Google Scholar 

  4. Middleton FA, Peng L, Lewis DA, Levitt P, Mirnics K. Altered expression of 14-3-3 in the prefrontal cortex of subjects with schizophrenia. Neuropsychopharmacology. 2005;30:974–83.

    CAS  PubMed  Article  Google Scholar 

  5. English JA, Pennington K, Dunn MJ, Cotter DR. The neuroproteomics of schizophrenia. Biol Psychiatry. 2011;69:163–72.

    CAS  PubMed  Article  Google Scholar 

  6. Pagan C, Delorme R, Callebert J, Goubran-Botros H, Amsellem F, Drouot X, et al. The serotonin-N-acetylserotonin-melatonin pathway as a biomarker for autism spectrum disorders. Transl Psychiatry. 2014;4:e479.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. Pagan C, Goubran-Botros H, Delorme R, Benabou M, Lemière N, Murray K, et al. Disruption of melatonin synthesis is associated with impaired 14-3-3 and miR-451 levels in patients with autism spectrum disorders. Sci Rep. 2017;7:2096.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  8. Cornell B, Toyo-oka K. 14-3-3 proteins in brain development: neurogenesis, neuronal migration and neuromorphogenesis. Front Mol Neurosci. 2017;10:318.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  9. Xu X, Jaehne EJ, Greenberg Z, McCarthy P, Saleh E, Parish CL, et al. 14-3-3ζ deficient mice in the BALB/c background display behavioural and anatomical defects associated with neurodevelopmental disorders. Sci Rep. 2015;5:12434.

    PubMed  PubMed Central  Article  Google Scholar 

  10. Toyo-Oka K, Wachi T, Hunt RF, Baraban SC, Taya S, Ramshaw H, et al. 14-3-3Ε and Ζ regulate neurogenesis and differentiation of neuronal progenitor cells in the developing brain. J Neurosci. 2014;34:12168–81.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Jaehne EJ, Ramshaw H, Xu X, Saleh E, Clark SR, Schubert KO, et al. In-vivo administration of clozapine affects behaviour but does not reverse dendritic spine deficits in the 14-3-3ζ KO mouse model of schizophrenia-like disorders. Pharm Biochem Behav. 2015;138:1–8.

    CAS  Article  Google Scholar 

  12. Cheah PS, Ramshaw HS, Thomas PQ, Toyo-Oka K, Xu X, Martin S, et al. Neurodevelopmental and neuropsychiatric behaviour defects arise from 14-3-3ζ deficiency. Mol Psychiatry. 2012;17:451–66.

    CAS  PubMed  Article  Google Scholar 

  13. Kalueff AV, Stewart AM, Gerlai R. Zebrafish as an emerging model for studying complex brain disorders. Trends Pharm Sci. 2014;35:63–75.

    CAS  PubMed  Article  Google Scholar 

  14. Norton W. Towards developmental models of psychiatric disorders in zebrafish. Front Neural Circuits. 2013;7:79.

    PubMed  PubMed Central  Article  Google Scholar 

  15. Vaz R, Hofmeister W, Lindstrand A. Zebrafish models of neurodevelopmental disorders: limitations and benefits of current tools and techniques. Int J Mol Sci. 2019;20:1296.

    CAS  PubMed Central  Article  Google Scholar 

  16. Kozol RA, Abrams AJ, James DM, Buglo E, Yan Q, Dallman JE. Function over form: Modeling groups of inherited neurological conditions in zebrafish. Front Mol Neurosci. 2016;9:55.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  17. Ahrens MB, Orger MB, Robson DN, Li JM, Keller PJ. Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat Methods. 2013;10:413–20.

    CAS  PubMed  Article  Google Scholar 

  18. Vanwalleghem GC, Ahrens MB, Scott EK. Integrative whole-brain neuroscience in larval zebrafish. Curr Opin Neurobiol. 2018;50:136–45.

    CAS  PubMed  Article  Google Scholar 

  19. Olarte OE, Andilla J, Gualda EJ, Loza-Alvarez P. Light-sheet microscopy: a tutorial. Adv Opt Photonics. 2018;10:111–79.

    Article  Google Scholar 

  20. Giovannucci A, Friedrich J, Gunn P, Kalfon J, Brown BL, Koay SA, et al. CaImAn an open-source tool for scalable calcium imaging data analysis. Elife. 2019;8:e38173.

    PubMed  PubMed Central  Article  Google Scholar 

  21. Orlandi JG, Fernández-García S, Comella-Bolla A, Masana M, Barriga GG-D, Yaghoubi M, et al. NETCAL: an interactive platform for large-scale, NETwork and population dynamics analysis of CALcium imaging recordings (7.0.0 Open Beta). Zenodo. 2017: https://doi.org/10.5281/zenodo.1119026.

  22. Young AMJ. Increased extracellular dopamine in nucleus accumbens in response to unconditioned and conditioned aversive stimuli: Studies using 1 min microdialysis in rats. J Neurosci Methods. 2004;138:57–63.

    CAS  PubMed  Article  Google Scholar 

  23. Ahn AH, Dziennis S, Hawkes R, Herrup K. The cloning of zebrin II reveals its identity with aldolase C. Development. 1994;120:2081–90.

    CAS  PubMed  Article  Google Scholar 

  24. McFarland KA, Topczewska JM, Weidinger G, Dorsky RI, Appel B. Hh and Wnt signaling regulate formation of olig2+ neurons in the zebrafish cerebellum. Dev Biol. 2008;318:162–71.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. Biechl D, Dorigo A, Köster RW, Grothe B, Wullimann MF. Eppur Si muove: Evidence for an external granular layer and possibly transit amplification in the teleostean cerebellum. Front Neuroanat. 2016;10:49.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  26. Kai W, Kunwar KCJ, Elisa DV, Sunil P, Norbert G, Anne D, et al. Cysteine modification by ebselen reduces the stability and cellular levels of 14-3-3 proteins. Mol Pharmacol. 2021;100:155–69.

    Article  CAS  Google Scholar 

  27. Molnár Z, Luhmann HJ, Kanold PO. Transient cortical circuits match spontaneous and sensory-driven activity during development. Science. 2020;370:eabb2153.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  28. Marachlian E, Avitan L, Goodhill GJ, Sumbre G. Principles of functional circuit connectivity: Insights from spontaneous activity in the zebrafish optic tectum. Front Neural Circuits. 2018;12:46.

    PubMed  PubMed Central  Article  Google Scholar 

  29. Avitan L, Pujic Z, Mölter J, Van De Poll M, Sun B, Teng H, et al. Spontaneous activity in the zebrafish tectum reorganizes over development and is influenced by visual experience. Curr Biol. 2017;27:2407–24.e4.

    CAS  PubMed  Article  Google Scholar 

  30. Momose-Sato Y, Sato K. Development of spontaneous activity in the avian hindbrain. Front Neural Circuits. 2016;10:63.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  31. Aitken A. 14-3-3 proteins: a historic overview. Semin Cancer Biol. 2006;16:162–72.

    CAS  PubMed  Article  Google Scholar 

  32. Vaswani M, Linda FK, Ramesh S. Role of selective serotonin reuptake inhibitors in psychiatric disorders: a comprehensive review. Prog Neuro-Psychopharmacol. Biol Psychiatry. 2003;27:85–102.

    CAS  Article  Google Scholar 

  33. Millan MJ, Maiofiss L, Cussac D, Audinot V, Boutin JA, Newman-Tancredi A. Differential actions of antiparkinson agents at multiple classes of monoaminergic receptor. I. A multivariate analysis of the binding profiles of 14 drugs at 21 native and cloned human receptor subtypes. J Pharm Exp Ther. 2002;303:791–804.

    CAS  Article  Google Scholar 

  34. Packer A. Neocortical neurogenesis and the etiology of autism spectrum disorder. Neurosci Biobehav Rev. 2016;64:185–95.

    PubMed  Article  Google Scholar 

  35. Stoodley CJ. The cerebellum and neurodevelopmental disorders. Cerebellum. 2016;15:34–37.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. van der Heijden ME, Gill JS, Sillitoe RV. Abnormal cerebellar development in autism spectrum disorders. Dev Neurosci. 2021;43:181–90.

    PubMed  Article  CAS  Google Scholar 

  37. Fatemi SH, Halt AR, Realmuto G, Earle J, Kist DA, Thuras P, et al. Purkinje cell size is reduced in cerebellum of patients with autism. Cell Mol Neurobiol. 2002;22:171–5.

    PubMed  Article  Google Scholar 

  38. Palmen SJMC, van Engeland H, Hof PR, Schmitz C. Neuropathological findings in autism. Brain. 2004;127:2572–83.

    PubMed  Article  Google Scholar 

  39. Bailey A, Luthert P, Dean A, Harding B, Janota I, Montgomery M, et al. A clinicopathological study of autism. Brain. 1998;121:889–905.

    PubMed  Article  Google Scholar 

  40. Skefos J, Cummings C, Enzer K, Holiday J, Weed K, Levy E. et al. Regional alterations in Purkinje cell density. PLoS One. 2014;9:e81255

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  41. Tsai PT, Hull C, Chu Y, Greene-Colozzi E, Sadowski AR, Leech JM, et al. Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice. Nature. 2012;488:647–51.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Kirkby LA, Sack GS, Firl A, Feller MB. A role for correlated spontaneous activity in the assembly of neural circuits. Neuron. 2013;80:1129–44.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. Blanquie O, Yang JW, Kilb W, Sharopov S, Sinning A, Luhmann HJ. Electrical activity controls area-specific expression of neuronal apoptosis in the mouse developing cerebral cortex. Elife. 2017;6:e27696.

    PubMed  PubMed Central  Article  Google Scholar 

  44. O’Reilly C, Lewis JD, Elsabbagh M. Is functional brain connectivity atypical in autism? A systematic review of EEG and MEG studies. PLoS One. 2017;12:e0175870.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  45. Rane P, Cochran D, Hodge SM, Haselgrove C, Kennedy DN, Frazier JA. Connectivity in autism: a review of MRI connectivity studies. Harv Rev Psychiatry. 2015;23:223–44.

    PubMed  PubMed Central  Article  Google Scholar 

  46. Li X, Zhang K, He X, Zhou J, Jin C. Structural, functional, and molecular imaging of autism spectrum disorder. Neurosci Bull. 2021;37:1051–71.

    PubMed  Article  Google Scholar 

  47. Sheffield JM, Barch DM. Cognition and resting-state functional connectivity in schizophrenia. Neurosci Biobehav Rev. 2016;61:108–20.

    PubMed  Article  Google Scholar 

  48. Van Den Heuvel MP, Fornito A. Brain networks in schizophrenia. Neuropsychol Rev. 2014;24:32–48.

    PubMed  Article  Google Scholar 

  49. Wang J, Lou H, Pedersen CJ, Smith AD, Perez RG. 14-3-3ζ contributes to tyrosine hydroxylase activity in MN9D cells: Localization of dopamine regulatory proteins to mitochondria. J Biol Chem. 2009;284:14011–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. Kesby JP, Eyles DW, McGrath JJ, Scott JG. Dopamine, psychosis and schizophrenia: the widening gap between basic and clinical neuroscience. Transl Psychiatry. 2018;8:30.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. Pavǎl D. A dopamine hypothesis of autism spectrum disorder. Dev Neurosci. 2017;39:355–60.

    PubMed  Article  CAS  Google Scholar 

  52. Tripp G, Wickens JR. Neurobiology of ADHD. Neuropharmacology. 2009;57:579–89.

    CAS  PubMed  Article  Google Scholar 

  53. Zakzanis KK, Hansen KT. Dopamine D2 densities and the schizophrenic brain. Schizophr Res. 1998;32:201–6.

    CAS  PubMed  Article  Google Scholar 

  54. Kestler LP, Walker E, Vega EM. Dopamine receptors in the brains of schizophrenia patients: a meta-analysis of the findings. Behav Pharmacol. 2001;12:355–71.

    CAS  PubMed  Article  Google Scholar 

  55. Gabriele S, Sacco R, Persico AM. Blood serotonin levels in autism spectrum disorder: a systematic review and meta-analysis. Eur Neuropsychopharmacol. 2014;24:919–29.

    CAS  PubMed  Article  Google Scholar 

  56. Muck-Seler D, Pivac N, Mustapic M, Crncevic Z, Jakovljevic M, Sagud M. Platelet serotonin and plasma prolactin and cortisol in healthy, depressed and schizophrenic women. Psychiatry Res. 2004;127:217–26.

    CAS  PubMed  Article  Google Scholar 

  57. Ramshaw H, Xu X, Jaehne EJ, McCarthy P, Greenberg Z, Saleh E, et al. Locomotor hyperactivity in 14-3-3ζ KO mice is associated with dopamine transporter dysfunction. Transl Psychiatry. 2013;3:e327.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. Persico AM, Ricciardello A, Lamberti M, Turriziani L, Cucinotta F, Brogna C, et al. The pediatric psychopharmacology of autism spectrum disorder: a systematic review—Part I: the past and the present. Prog Neuro-Psychopharmacol. Biol Psychiatry. 2021;110:110326.

    CAS  Article  Google Scholar 

  59. Jacob SN, Nienborg H. Monoaminergic neuromodulation of sensory processing. Front Neural Circuits. 2018;12:51.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  60. Fernández M, Mollinedo-Gajate I, Peñagarikano O. Neural circuits for social cognition: implications for autism. Neuroscience. 2018;370:148–62.

    PubMed  Article  CAS  Google Scholar 

  61. Rademacher L, Schulte-Rüther M, Hanewald B, Lammertz S. Reward: from basic reinforcers to anticipation of social cues. Curr Top Behav Neurosci.2016;30:207–21.

    Article  Google Scholar 

  62. Franceschini A, Fattore L. Gender-specific approach in psychiatric diseases: because sex matters. Eur J Pharmacol. 2021;896:173895.

    CAS  PubMed  Article  Google Scholar 

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Acknowledgements

GCaMP6s albino zebrafish embryos were generated by the National Institute of Genetics (Japan) and obtained from Dr. Matt Parker from the University of Portsmouth, UK. The Tg(aldoca:gap43-Venus) line was obtained from Masahiko Hibi from the Bioscience and Biotechnology Center of Nagoya University, Japan. Tg(olig2:egfp)vu12 brains were obtained from the Center for Developmental Biology, UMR 5547 CNRS, Toulouse, France. Major financial support for this research was received by BC from the Spanish ‘Ministerio de Ciencia, Innovación y Universidades’ (RTI2018-100968-B-100, PID2021-1277760B-I100), the ‘Ministerio de Sanidad, Servicios Sociales e Igualdad/Plan Nacional Sobre Drogas’ (PNSD-2017I050 and PNSD-2020I042), ‘Generalitat de Catalunya/AGAUR’ (2017-SGR-738), ICREA Academia 2021, and the European Union H2020 Program [H2020/2014-2020] under grant agreements n° 667302 (CoCA) and Eat2beNICE (728018). EA-G was supported by the Ministerio de Economía y Competitividad (Spanish Government) and the EU H2020 programme (Eat2beNICE-728018). GC, EG and PL-A acknowledge financial support from the Spanish Ministerio de Economía y Competitividad (MINECO) through the “Severo Ochoa” programme for Centres of Excellence in R&D CEX2019-000910-S), MINECO/FEDER Ramon y Cajal programme (RYC-2015-17935); Laserlab-Europe EU-H2020 GA no. 871124, Fundació Privada Cellex, Fundación Mig-Puig and from the Generalitat de Catalunya through the CERCA programme. FA acknowledges financial support from the Spanish ‘Ministerio de Ciencia, Innovación y Universidades’ (PID2019-107738RB-I00, MICINN/FEDER) and SGR (2017SGR1255).

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NF-C, WHJN and BC conceived and coordinated the study, NF-C designed the experimental approaches for whole-brain imaging, WHJN designed the behavioural and pharmacological approaches and BC designed the genetic approaches. EA-G designed and conducted the whole-brain imaging and behavioural experiments and wrote the paper, EDV designed and conducted the CRISPR/Cas9, ISH, IHC, HPLC and behavioural experiments. AMJY contributed to the HPLC experiments. CH-U and JG-F carried out the phylogenetic analysis. MG-P contributed to the behavioural experiments. CA contributed to the statistical analysis of the behavioural tests. JGO designed the pipeline and methodology for the whole-brain imaging analyses, GC and EG conducted the whole-brain imaging recordings, EA-G analysed the imaging data, FA contributed to the whole-brain imaging analysis and PL-A supervised the whole-brain imaging recordings. All authors discussed and commented on the manuscript.

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Correspondence to Bru Cormand, William H. J. Norton or Noèlia Fernàndez-Castillo.

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Antón-Galindo, E., Dalla Vecchia, E., Orlandi, J.G. et al. Deficiency of the ywhaz gene, involved in neurodevelopmental disorders, alters brain activity and behaviour in zebrafish. Mol Psychiatry (2022). https://doi.org/10.1038/s41380-022-01577-9

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