Loss of sdhb in zebrafish larvae recapitulates human paraganglioma characteristics

in Endocrine-Related Cancer
View More View Less
  • 1 Department of Internal Medicine, Radboud University Medical Center, Nijmegen, the Netherlands
  • 2 Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, the Netherlands
  • 3 Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universitat Dresden, Dresden, Germany
  • 4 Department of Medicine ΙΙΙ, University Hospital Dresden, Dresden, Germany
  • 5 Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Utrecht, the Netherlands
  • 6 Division of Heart and Lungs, Department of Medical Physiology, UMC Utrecht, Utrecht, the Netherlands
  • 7 Department of Biomedical, Experimental and Clinical Sciences ‘Mario Serio’, University of Florence, Firenze, Italy
  • 8 Department of Pediatrics, Radboud Center for Mitochondrial Medicine, Translational Metabolic Laboratory, Radboud University Medical Center, Nijmegen, the Netherlands
  • 9 Department of Animal Ecology and Physiology, Institute for Water and Wetland Research, Radboud University, Nijmegen, the Netherlands
  • 10 Radboud University, Nijmegen, the Netherlands

Correspondence should be addressed to M Dona: margo.dona@radboudumc.nl
Restricted access

Pheochromocytomas and paragangliomas (PPGLs) caused by mutations in the B-subunit of the succinate dehydrogenase (SDHB) have the highest metastatic rate among PPGLs, and effective systemic therapy is lacking. To unravel underlying pathogenic mechanisms, and to evaluate therapeutic strategies, suitable in vivo models are needed. The available systemic Sdhb knock-out mice cannot model the human PPGL phenotype: heterozygous Sdhb mice lack a disease phenotype, and homozygous Sdhb mice are embryonically lethal. Using CRISPR/cas9 technology, we introduced a protein-truncating germline lesion into the zebrafish sdhb gene. Heterozygous sdhb mutants were viable and displayed no obvious morphological or developmental defects. Homozygous sdhb larvae were viable, but exhibited a decreased lifespan. Morphological analysis revealed incompletely or non-inflated swim bladders in homozygous sdhb mutants at day 6. Although no differences in number and ultrastructure of the mitochondria were observed. Clear defects in energy metabolism and swimming behavior were observed in homozygous sdhb mutant larvae. Functional and metabolomic analyses revealed decreased mitochondrial complex 2 activity and significant succinate accumulation in the homozygous sdhb mutant larvae, mimicking the metabolic effects observed in SDHB-associated PPGLs. This is the first study to present a vertebrate animal model that mimics metabolic effects of SDHB-associated PPGLs. This model will be useful in unraveling pathomechanisms behind SDHB-associated PPGLs. We can now study the metabolic effects of sdhb disruption during different developmental stages and develop screening assays to identify novel therapeutic targets in vivo. Besides oncological syndromes, our model might also be useful for pediatric mitochondrial disease caused by loss of the SDHB gene.

Supplementary Materials

    • Supplementary Figure 1. Alignment of amino acid sequences of human SDHB and the two candidate homologs in zebrafish, sdhb and SDHB. Probably damaging (black), possibly damaging (grey) and non-damaging (light grey) amino acid substitutions according to Polyphen are indicated. Identical, highly similar and somewhat similar amino acids are indicated by stars, two dots, and one dot, respectively. The arrow points at the substitution in zebrafish SDHB, which is also found in a paraganglioma patient.
    • Supplementary figure 2. Evolutionary conservation of SDHB amino acids. Multi-sequence alignment of SDHB protein sequences of human, mouse, and fish. A star and grey background indicate identical amino acids, two dots a highly similar amino acids, and one dot a somewhat similar amino acid. A black background indicates the presence of a different amino acid in zebrafish SDHB compared to all other species. The black-outlined boxes indicate extra stretches or missing stretches of sequence in zebrafish SDHB compared to all other species.
    • Supplementary Figure 3. Hematoxylin and eosin staining. No major differences were found in heart, liver and kidney between both sdhb mutants and wild-type siblings at 6dpf (A-B). Inflammation process is observed in the intestine of homozygous sdhb larvae, characterized by shorter primary and secondary mucosal folds pointed by the black ellipse and asterisk and an increase number of goblet cells indicated by black arrows (C-D). Scalebar, 200µm (A, B) and 50µm (C,D).
    • Supplementary Figure 4. Electron microscopy images of 6dpf old larvae. No differences were observed in mitochondrial morphology in muscle (A-C) and brain (A’-C’). Minimum of n=3 per genotype. Scale bars: 500nm. M: Mitochondria.
    • Supplementary Table 1: Primer oligonucleotides sequences for gene expression analysis.

 

Society for Endocrinology

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 682 682 70
Full Text Views 83 83 4
PDF Downloads 70 70 7
  • Alston CL, Davison JE, Meloni F, Van Der Westhuizen FH, He L, Hornig-Do HT, Peet AC, Gissen P, Goffrini P & Ferrero I et al. 2012 Recessive germline SDHA and SDHB mutations causing leukodystrophy and isolated mitochondrial complex II deficiency. Journal of Medical Genetics 49 5695 77. (https://doi.org/10.1136/jmedgenet-2012-101146)

    • Search Google Scholar
    • Export Citation
  • Briere JJ, Favier J, El Ghouzzi V, Djouadi F, Benit P, Gimenez AP & Rustin P 2005 Succinate dehydrogenase deficiency in human. Cellular and Molecular Life Sciences 62 23172 32 4. (https://doi.org/10.1007/s00018-005-5237-6)

    • Search Google Scholar
    • Export Citation
  • Burnichon N, Rohmer V, Amar L, Herman P, Leboulleux S, Darrouzet V, Niccoli P, Gaillard D, Chabrier G & Chabolle F et al. 2009 The succinate dehydrogenase genetic testing in a large prospective series of patients with paragangliomas. Journal of Clinical Endocrinology and Metabolism 94 28172 82 7. (https://doi.org/10.1210/jc.2008-2504)

    • Search Google Scholar
    • Export Citation
  • Carradice D & Lieschke GJ 2008 Zebrafish in hematology: sushi or science? Blood 111 333133 42. (https://doi.org/10.1182/blood-2007-10-052761)

    • Search Google Scholar
    • Export Citation
  • Cascon A, Remacha L, Calsina B & Robledo M 2019 Pheochromocytomas and paragangliomas: bypassing cellular respiration. Cancers 11 683. (https://doi.org/10.3390/cancers11050683)

    • Search Google Scholar
    • Export Citation
  • Cervera AM, Apostolova N, Crespo FL, Mata M & Mccreath KJ 2008 Cells silenced for SDHB expression display characteristic features of the tumor phenotype. Cancer Research 68 405840 67. (https://doi.org/10.1158/0008-5472.CAN-07-5580)

    • Search Google Scholar
    • Export Citation
  • Connors J, Dawe N & Van Limbergen J 2018 The role of succinate in the regulation of intestinal inflammation. Nutrients 11 25. (https://doi.org/10.3390/nu11010025)

    • Search Google Scholar
    • Export Citation
  • Despic V & Neugebauer KM 2018 RNA tales – how embryos read and discard messages from mom. Journal of Cell Science 131 jcs.201996. (https://doi.org/10.1242/jcs.201996)

    • Search Google Scholar
    • Export Citation
  • Favier J, Plouin PF, Corvol P & Gasc JM 2002 Angiogenesis and vascular architecture in pheochromocytomas: distinctive traits in malignant tumors. American Journal of Pathology 161 123512 46. (https://doi.org/10.1016/S0002-9440(10)64400-8)

    • Search Google Scholar
    • Export Citation
  • Favier J, Briere JJ, Burnichon N, Riviere J, Vescovo L, Benit P, Giscos-Douriez I, De Reynies A, Bertherat J & Badoual C et al. 2009 The Warburg effect is genetically determined in inherited pheochromocytomas. PLoS ONE 4 e7094. (https://doi.org/10.1371/journal.pone.0007094)

    • Search Google Scholar
    • Export Citation
  • Feitsma H & Cuppen E 2008 Zebrafish as a cancer model. Molecular Cancer Research 6 6856 94. (https://doi.org/10.1158/1541-7786.MCR-07-2167)

  • Frezza C, Zheng L, Folger O, Rajagopalan KN, Mackenzie ED, Jerby L, Micaroni M, Chaneton B, Adam J & Hedley A et al. 2011 Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Nature 477 22522 8. (https://doi.org/10.1038/nature10363)

    • Search Google Scholar
    • Export Citation
  • Gimenez-Roqueplo AP, Favier J, Rustin P, Rieubland C, Kerlan V, Plouin PF, Rotig A & Jeunemaitre X 2002 Functional consequences of a SDHB gene mutation in an apparently sporadic pheochromocytoma. Journal of Clinical Endocrinology and Metabolism 87 47714 774. (https://doi.org/10.1210/jc.2002-020525)

    • Search Google Scholar
    • Export Citation
  • Gottlieb E & Tomlinson IPM 2005 Mitochondrial tumour suppressors: a genetic and biochemical update. Nature Reviews Cancer 5 8578 66. (https://doi.org/10.1038/nrc1737)

    • Search Google Scholar
    • Export Citation
  • Gronborg S, Darin N, Miranda MJ, Damgaard B, Cayuela JA, Oldfors A, Kollberg G, Hansen TVO, Ravn K & Wibrand F et al. 2016 Leukoencephalopathy due to complex II deficiency and bi-allelic SDHB mutations: further cases and implications for genetic counselling. JIMD Reports 33 6977. (https://doi.org/10.1007/8904_2016_582)

    • Search Google Scholar
    • Export Citation
  • Harvey SA, Sealy I, Kettleborough R, Fenyes F, White R, Stemple D & Smith JC 2013 Identification of the zebrafish maternal and paternal transcriptomes. Development 140 270327 10. (https://doi.org/10.1242/dev.095091)

    • Search Google Scholar
    • Export Citation
  • Hruscha A, Krawitz P, Rechenberg A, Heinrich V, Hecht J, Haass C & Schmid B 2013 Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. Development 140 4982498 7. (https://doi.org/10.1242/dev.099085)

    • Search Google Scholar
    • Export Citation
  • Kimmel CB, Ballard WW, Kimmel SR, Ullmann B & Schilling TF 1995 Stages of embryonic development of the zebrafish. Developmental Dynamics 203 253310. (https://doi.org/10.1002/aja.1002030302)

    • Search Google Scholar
    • Export Citation
  • King KS, Prodanov T, Kantorovich V, Fojo T, Hewitt JK, Zacharin M, Wesley R, Lodish M, Raygada M & Gimenez-Roqueplo AP et al. 2011 Metastatic pheochromocytoma/paraganglioma related to primary tumor development in childhood or adolescence: significant link to SDHB mutations. Journal of Clinical Oncology 29 41374 14 2. (https://doi.org/10.1200/JCO.2011.34.6353)

    • Search Google Scholar
    • Export Citation
  • Kitazawa S, Ebara S, Ando A, Baba Y, Satomi Y, Soga T & Hara T 2017 Succinate dehydrogenase B-deficient cancer cells are highly sensitive to bromodomain and extra-terminal inhibitors. Oncotarget 8 2892228938. (https://doi.org/10.18632/oncotarget.15959)

    • Search Google Scholar
    • Export Citation
  • Kluckova K & Tennant DA 2018 Metabolic implications of hypoxia and pseudohypoxia in pheochromocytoma and paraganglioma. Cell and Tissue Research 372 367378. (https://doi.org/10.1007/s00441-018-2801-6)

    • Search Google Scholar
    • Export Citation
  • Kruse F, Junker JP, Van Oudenaarden A & Bakkers J 2016 Tomo-seq: a method to obtain genome-wide expression data with spatial resolution. Methods in Cell Biology 135 299307. (https://doi.org/10.1016/bs.mcb.2016.01.006)

    • Search Google Scholar
    • Export Citation
  • Lam AK 2017 Update on adrenal tumours in 2017 World Health Organization (WHO) of endocrine tumours. Endocrine Pathology 28 213227. (https://doi.org/10.1007/s12022-017-9484-5)

    • Search Google Scholar
    • Export Citation
  • Lepoutre-Lussey C, Thibault C, Buffet A, Morin A, Badoual C, Benit P, Rustin P, Ottolenghi C, Janin M & Castro-Vega LJ et al. 2016 From Nf1 to Sdhb knockout: successes and failures in the quest for animal models of pheochromocytoma. Molecular and Cellular Endocrinology 421 404 8. (https://doi.org/10.1016/j.mce.2015.06.027)

    • Search Google Scholar
    • Export Citation
  • Lindsey BW, Smith FM & Croll RP 2010 From inflation to flotation: contribution of the swimbladder to whole-body density and swimming depth during development of the zebrafish (Danio rerio). Zebrafish 7 8596. (https://doi.org/10.1089/zeb.2009.0616)

    • Search Google Scholar
    • Export Citation
  • Liu Y, Pang Y, Zhu B, Uher O, Caisova V, Huynh TT, Taieb D, Hadrava Vanova K, Ghayee HK & Neuzil J et al. 2020 Therapeutic targeting of SDHB-mutated pheochromocytoma/paraganglioma with pharmacologic ascorbic acid. Clinical Cancer Research 26 38683880. (https://doi.org/10.1158/1078-0432.CCR-19-2335)

    • Search Google Scholar
    • Export Citation
  • Lopez-Jimenez E, Gomez-Lopez G, Leandro-Garcia LJ, Munoz I, Schiavi F, Montero-Conde C, De Cubas AA, Ramires R, Landa I & Leskela S et al. 2010 Research resource: transcriptional profiling reveals different pseudohypoxic signatures in SDHB and VHL-related pheochromocytomas. Molecular Endocrinology 24 238223 91. (https://doi.org/10.1210/me.2010-0256)

    • Search Google Scholar
    • Export Citation
  • Loriot C, Domingues M, Berger A, Menara M, Ruel M, Morin A, Castro-Vega LJ, Letouze É, Martinelli C & Bemelmans AP et al. 2015 Deciphering the molecular basis of invasiveness in Sdhb-deficient cells. Oncotarget 6 32955329 65. (https://doi.org/10.18632/oncotarget.5106)

    • Search Google Scholar
    • Export Citation
  • Lussey-Lepoutre C, Hollinshead KER, Ludwig C, Menara M, Morin A, Castro-Vega LJ, Parker SJ, Janin M, Martinelli C & Ottolenghi C et al. 2015 Loss of succinate dehydrogenase activity results in dependency on pyruvate carboxylation for cellular anabolism. Nature Communications 6 8784. (https://doi.org/10.1038/ncomms9784)

    • Search Google Scholar
    • Export Citation
  • Lussey-Lepoutre C, Buffet A, Morin A, Goncalves J & Favier J 2018 Rodent models of pheochromocytoma, parallels in rodent and human tumorigenesis. Cell and Tissue Research 372 379392. (https://doi.org/10.1007/s00441-018-2797-y)

    • Search Google Scholar
    • Export Citation
  • Macrae CA & Peterson RT 2015 Zebrafish as tools for drug discovery. Nature Reviews Drug Discovery 14 7217 31. (https://doi.org/10.1038/nrd4627)

    • Search Google Scholar
    • Export Citation
  • Moosavi B, Zhu XL, Yang WC & Yang GF 2020 Molecular pathogenesis of tumorigenesis caused by succinate dehydrogenase defect. European Journal of Cell Biology 99 151057. (https://doi.org/10.1016/j.ejcb.2019.151057)

    • Search Google Scholar
    • Export Citation
  • Olson SW, Yoon S, Baker T, Prince LK, Oliver D & Abbott KC 2016 Longitudinal plasma metanephrines preceding pheochromocytoma diagnosis: a retrospective case-control serum repository study. European Journal of Endocrinology 174 2892 95. (https://doi.org/10.1530/EJE-15-0651)

    • Search Google Scholar
    • Export Citation
  • Paffett-Lugassy N, Hsia N, Fraenkel PG, Paw B, Leshinsky I, Barut B, Bahary N, Caro J, Handin R & Zon LI 2007 Functional conservation of erythropoietin signaling in zebrafish. Blood 110 27182 72 6. (https://doi.org/10.1182/blood-2006-04-016535)

    • Search Google Scholar
    • Export Citation
  • Pollard PJ, Briere JJ, Alam NA, Barwell J, Barclay E, Wortham NC, Hunt T, Mitchell M, Olpin S & Moat SJ et al. 2005 Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations. Human Molecular Genetics 14 2231223 9. (https://doi.org/10.1093/hmg/ddi227)

    • Search Google Scholar
    • Export Citation
  • Powers JF, Cochran B, Baleja JD, Sikes HD, Pattison AD, Zhang X, Lomakin I, Shepard-Barry A, Pacak K & Moon SJ et al. 2020 A xenograft and cell line model of SDH-deficient pheochromocytoma derived from Sdhb+/- rats. Endocrine-Related Cancer 27 337354. (https://doi.org/10.1530/ERC-19-0474)

    • Search Google Scholar
    • Export Citation
  • Rao JU, Engelke UF, Rodenburg RJ, Wevers RA, Pacak K, Eisenhofer G, Qin N, Kusters B, Goudswaard AG & Lenders JW et al. 2013 Genotype-specific abnormalities in mitochondrial function associate with distinct profiles of energy metabolism and catecholamine content in pheochromocytoma and paraganglioma. Clinical Cancer Research 19 378737 95. (https://doi.org/10.1158/1078-0432.CCR-12-3922)

    • Search Google Scholar
    • Export Citation
  • Richter S, Peitzsch M, Rapizzi E, Lenders JW, Qin N, De Cubas AA, Schiavi F, Rao JU, Beuschlein F & Quinkler M et al. 2014 Krebs cycle metabolite profiling for identification and stratification of pheochromocytomas/paragangliomas due to succinate dehydrogenase deficiency. Journal of Clinical Endocrinology and Metabolism 99 390339 11. (https://doi.org/10.1210/jc.2014-2151)

    • Search Google Scholar
    • Export Citation
  • Richter S, D’antongiovanni V, Martinelli S, Bechmann N, Riverso M, Poitz DM, Pacak K, Eisenhofer G, Mannelli M & Rapizzi E 2018 Primary fibroblast co-culture stimulates growth and metabolism in Sdhb-impaired mouse pheochromocytoma MTT cells. Cell and Tissue Research 374 473485. (https://doi.org/10.1007/s00441-018-2907-x)

    • Search Google Scholar
    • Export Citation
  • Richter S, Gieldon L, Pang Y, Peitzsch M, Huynh T, Leton R, Viana B, Ercolino T, Mangelis A & Rapizzi E et al. 2019 Metabolome-guided genomics to identify pathogenic variants in isocitrate dehydrogenase, fumarate hydratase, and succinate dehydrogenase genes in pheochromocytoma and paraganglioma. Genetics in Medicine 21 705717. (https://doi.org/10.1038/s41436-018-0106-5)

    • Search Google Scholar
    • Export Citation
  • Rijken JA, Niemeijer ND, Jonker MA, Eijkelenkamp K, Jansen JC, Van Berkel A, Timmers HJLM, Kunst HPM, Bisschop PHLT & Kerstens MN et al. 2018 The penetrance of paraganglioma and pheochromocytoma in SDHB germline mutation carriers. Clinical Genetics 93 6066. (https://doi.org/10.1111/cge.13055)

    • Search Google Scholar
    • Export Citation
  • Rodenburg RJT 2011 Biochemical diagnosis of mitochondrial disorders. Journal of Inherited Metabolic Disease 34 2832 92. (https://doi.org/10.1007/s10545-010-9081-y)

    • Search Google Scholar
    • Export Citation
  • Sander JD, Zaback P, Joung JK, Voytas DF & Dobbs D 2007 Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic Acids Research 35 W599W 605. (https://doi.org/10.1093/nar/gkm349)

    • Search Google Scholar
    • Export Citation
  • Selak MA, Armour SM, Mackenzie ED, Boulahbel H, Watson DG, Mansfield KD, Pan Y, Simon MC, Thompson CB & Gottlieb E 2005 Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase. Cancer Cell 7 7785. (https://doi.org/10.1016/j.ccr.2004.11.022)

    • Search Google Scholar
    • Export Citation
  • Smits AH, Ziebell F, Joberty G, Zinn N, Mueller WF, Clauder-Munster S, Eberhard D, Falth Savitski M, Grandi P & Jakob P et al. 2019 Biological plasticity rescues target activity in CRISPR knock outs. Nature Methods 16 10871093. (https://doi.org/10.1038/s41592-019-0614-5)

    • Search Google Scholar
    • Export Citation
  • Sulkowski PL, Sundaram RK, Oeck S, Corso CD, Liu Y, Noorbakhsh S, Niger M, Boeke M, Ueno D & Kalathil AN 2018 Krebs-cycle-deficient hereditary cancer syndromes are defined by defects in homologous-recombination DNA repair. Nature Genetics 50 108 6109 2. (https://doi.org/10.1038/s41588-018-0170-4)

    • Search Google Scholar
    • Export Citation
  • Timmers HJLM, Kozupa A, Eisenhofer G, Raygada M, Adams KT, Solis D, Lenders JWM & Pacak K 2007 Clinical presentations, biochemical phenotypes, and genotype-phenotype correlations in patients with succinate dehydrogenase subunit B-associated pheochromocytomas and paragangliomas. Journal of Clinical Endocrinology and Metabolism 92 7797 86. (https://doi.org/10.1210/jc.2006-2315)

    • Search Google Scholar
    • Export Citation
  • Van Rooijen E, Voest EE, Logister I, Korving J, Schwerte T, Schulte-Merker S, Giles RH & Van Eeden FJ 2009 Zebrafish mutants in the von Hippel-Lindau tumor suppressor display a hypoxic response and recapitulate key aspects of Chuvash polycythemia. Blood 113 64496 46 0. (https://doi.org/10.1182/blood-2008-07-167890)

    • Search Google Scholar
    • Export Citation
  • Yang MJ, Cheng ZX, Jiang M, Zeng ZH, Peng B, Peng XX & Li H 2018 Boosted TCA cycle enhances survival of zebrafish to Vibrio alginolyticus infection. Virulence 9 634644. (https://doi.org/10.1080/21505594.2017.1423188)

    • Search Google Scholar
    • Export Citation