Abstract
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.
Introduction
The succinate dehydrogenase (SDH) complex located in the inner mitochondrial membrane has an essential and unique dual role in the energy-production in eukaryotic cells. It is involved in both the oxidation of succinate to fumarate in the tricarboxylic acid (TCA)-cycle and the reduction of ubiquinones (co-enzyme Q) in the aerobic electron transfer chain, thus contributing to ATP production by oxidative phosphorylation (Gottlieb & Tomlinson 2005). The SDH complex is composed of four subunits (SDHA–D), that together with four assembly factor genes (SDHADF1, SDHAF2, SDHADF3 and SDHADF4), form the mitochondrial complex 2. Since complex 2 is involved in both the electron transport chain and TCA-cycle, defects lead to a variety of disease phenotypes ranging from severe neuromuscular disorders to different types of cancer.
In humans, homozygous or compound heterozygous SDH germline mutations, causing isolated complex 2 deficiencies, lead to congenital hypotonia and leukoencephalopathy and are often lethal (Alston et al. 2012, Gronborg et al. 2016). Isolated deficiency of complex 2 is a rare condition, accounting for an estimated 2% of respiratory chain deficiencies (Briere et al. 2005). Carriers of a heterozygous SDH mutation are at risk of developing pheochromocytomas and paragangliomas (PPGLs), although usually a second hit in the form of a somatic mutation in the unaffected SDHB allele is required for the development of PPGLs. PPGLs are rare, catecholamine-producing, neuroendocrine tumors that arise from the adrenal medulla or extra-adrenal paraganglia (Lam 2017). Hyperproduction of catecholamines by these chromaffin tumor cells typically results in symptoms that mimic sympathetic activation, including hypertension, tachycardia, headache, and increased sweating. PPGLs occur in up to eight per million persons per year. Although in the majority of cases PPGLs follow a benign course, at least 15% develop into metastatic disease (King et al. 2011). Currently, no effective treatment is available. SDHB mutations are of particular interest, because they represent the most common hereditary form of PPGL and are associated with the highest risk of malignancy. Germline SDHB mutations are encountered in 30–48% of patients with metastatic PPGLs and predispose to a malignant course in 37–97% of cases, in contrast to 10% for non-SDHB PPGLs (Timmers et al. 2007, Burnichon et al. 2009). At present, it is unknown why SDHB mutations specifically predispose to the malignant phenotype. Besides PPGLs, carriers of SDHB mutations are also at risk to develop renal cell carcinoma, gastrointestinal stromal tumors, pituitary adenoma and pancreatic neuroendocrine tumors (Lam 2017).
Due to the dual role of SDH in the TCA-cycle and in mitochondrial electron transport, SDH subunit mutations cause dysfunction of oxidative phosphorylation and hence increased production of reactive oxygen species (ROS), severely affecting cellular energy metabolism (Cascon et al. 2019, Moosavi et al. 2020). On the other hand, increased tissue succinate levels determined in SDH-related PPGLs (Pollard et al. 2005, Rao et al. 2013, Richter et al. 2014) act as an oncometabolite with broad cellular effects. Succinate inhibits prolyl hydroxylases (PHDs), resulting in stabilization of HIF1/2α transcription factors (Favier et al. 2002, 2009, Selak et al. 2005, Lopez-Jimenez et al. 2010). Increased HIF-2α levels induce the expression of genes that contain hypoxia-responsive elements; their activation results in tumor progression via various signaling pathways. After binding its transcription factor partner HIF-β/ARNT, HIF-2α activates transcription of angiogenic and proliferative genes as well as genes involved in metabolism, leading to the so-called Warburg effect (Kluckova & Tennant 2018). The Warburg effect entails a shift of metabolism from oxidative phosphorylation to aerobic glycolysis, as exemplified in PPGL by increased lactate dehydrogenase (LDHA) expression and increased blood lactate levels (Gimenez-Roqueplo et al. 2002, Favier et al. 2009, Frezza et al. 2011). Other pathophysiological mechanisms proposed to play a role in succinate-dependent tumorigenesis are epigenetic dysregulation via altered DNA and histone methylation profiles (Cascon et al. 2019), and decreased DNA repair via suppression of the homologous recombination pathway (Sulkowski et al. 2018).
To develop targeted therapies for SDHB-related PPGLs, better understanding of the etiology and pathophysiology is needed. So far, the search for a suitable animal model for SDHB-associated PPGLs was discouraging (Lepoutre-Lussey et al. 2016, Lussey-Lepoutre et al. 2018). Graft models using mouse or rat cells are currently the best available alternatives (Liu et al. 2020, Powers et al. 2020). Available in vivo mouse models with SDH disruption have not produced the desired outcomes (Lepoutre-Lussey et al. 2016). Similar to the human situation, mice carrying one inactivated Sdhb gene do not develop a disease phenotype, whereas homozygous and thus complete inactivation of Sdhb results in embryonic lethality. Heterozygous mouse Sdhb mutants showed no changes in sympathic and cardiovascular phenotype, nor did they showed any macroscopic or histological abnormalities of the main organs and (para)sympathetic tissues, such as the adrenal medulla and the carotic body, from which PPGLs would typically originate (Lepoutre-Lussey et al. 2016). In fact no SDHB-related knockout mouse model ever developed PPGLs. In line with these observations, conditional knock-out mice for the Von Hippel Lindau (VHL) gene, another PPGL susceptibility gene involved in HIF signaling, failed to mimic the human diseased phenotype. In contrast to Vhl knock-out mice, however, homozygous vhl mutant zebrafish did develop key aspects of the human disease condition, including polycythemia and severe neovascularization, a prominent effect of VHL-related tumors (Van Rooijen et al. 2009). Importantly, the zebrafish HIF-pathway, vascular and hematopoietic systems show remarkable functional conservation compared to humans (Paffett-Lugassy et al. 2007, Carradice & Lieschke 2008). Combined with the ease of genetic manipulation, the zebrafish’ translucency, ideal to image progression of tumor growth, and its potential for high-throughput drug screening assays as demonstrated for several tumor types (Feitsma & Cuppen 2008, Hruscha et al. 2013), the zebrafish constitutes a valid and powerful model for PPGL studies. Here, we generated and characterized a sdhb zebrafish transgene to study succinate dehydrogenase subunit B function, which is at the basis of pathomechanisms behind SDHB-associated tumorigenesis.
Materials and methods
Zebrafish husbandry
Experimental procedures were conducted in accordance with institutional guidelines and National and European laws. Ethical approval of the experiments was granted by Radboud University’s Institutional Animal Care and Use Committee (IACUC, application number RU-DEC 2015-0098). Wild type adult Oregon AB* zebrafish (Danio rerio) were used. Eggs were obtained from natural spawning. Larvae were maintained and raised by standard methods (Kimmel et al. 1995).
Bioinformatic analysis
Several online tools were used for bioinformatics analyses. In search for orthologues, we performed BLAST searches (https://blast.ncbi.nlm.nih.gov). For more detailed information on the zebrafish candidate sdhb orthologues, we gathered information from Zfin (http://zfin.org:; Gene-ID:030131-8005) and Ensembl.org; ENSDARG00000075768 and ENSDARG00000092713. Pairwise protein alignments were performed with the EMBOSS Needle tool (http://www.ebi.ac.uk/Tools/psa/emboss_needle/). Multi-sequence alignments were executed with the ClustalW tool (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Protein domains were identified with Interpro (https://www.ebi.ac.uk/interpro/). Expression data of sdhb was gathered from the Bgee database (http://bgee.org:; Gene-ID:ENSDARG00000075768), and the search engine from the research group of Jeroen Bakkers (Hubrecht Institute, The Netherlands) based on their tomoseq data (http://zebrafish.genomes.nl/tomoseq/Junker2014; Gene-ID:ENSDARG00000075768). Polyphen was used to predict the impact of amino acid substitutions on protein function (http://genetics.bwh.harvard.edu/pph2/).
CRISPR/cas9 design and microinjection
For the sdhbrmc200 allele, oligos to generate guide RNAs were designed using the ZiFiT targeter software (Sander et al. 2007); gRNA with targeting sequence 5’-GTTCACCGGCCCGCCGTCA-3’ was ordered from Sigma Aldrich. Zebrafish eggs at the 1-cell stage were injected with 1 nl of a mixture containing (150 ng/μL) gRNA, (300 ng/μL) cas9 protein (PNA Bio, CPO1) in water in combination with 0.05% (v/v) phenol red using a Pneumatic PicoPump pv280 (World Precision Instruments). After injection, embryos were raised at 28.5°C with a 14 h light:10 h darkness cycle in E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4), supplemented with 0.1% (v/v) methylene blue. At 60 h post-fertilization, the injected embryos were analyzed for the presence of desired mutational events and further raised when mutations were detected. Three-month-old fish (F0 founder fish) were selected via finclip for the 13 bp frameshift mutation at the sdhb exon1–intron1 boundary (c. 57_63 + 64_69del; p.Val20argfsTer43) and crossed to generate F1 heterozygous carriers. The adult F1–F3 heterozygous sdhbrmc200 carriers were crossed to generate F2–4 larvae which were used for experiments.
Genotyping
Larvae were briefly anesthetized in 2-phenoxyethanol (0.1%, v/v). Genomic DNA was isolated by finclipping the most anterior 0.5 mm of a tail fin with a small knife (Sharpoint 5 mm depth 15 degrees stab), or when the larvae was not required for further experiments, the whole 5 dpf larva was used to obtain genomic DNA. Either the fin was placed in 10 µL lysis buffer (40 mM NaOH, 0.2 mM EDTA) or the complete larvae was placed in 75 µL lysis buffer, subsequently followed by incubation at 95°C for 30 min. The isolated genomic DNA was used as PCR template. Primer sequences used for amplification of sdhb exon 1 are 5’-CATGGCGGCTGTGTGTTTCT-3’ (fw) and 5’-ACGGGTTATTGATGAGCGGTG-3’ (rv). As input 15.1 µL water, 0.2 µL Dreamtaq (5 U/µL), 0.5µL dNTPs (10 µM), 1 µL fw primer (10 µM), 1µL rv primer (10 µM), 2 µL PCR buffer and 1 µL isolated genomic DNA for the entire larvae and 2 µL of isolated genomic DNA of a tail fin was used. The cycling conditions were as follows: 94°C 2 min, 35 cycles of 94˚C 15 s, 58˚C 30 s, 72˚C 30 s and 72˚C 2 min. The obtained amplicons (WT: 123 basepairs (bp); heterozygous mutant: 123bp and 110bp and homozygous mutant: 110bp) were visualized on a 4% agarose gel and verified by Sanger sequence.
Behavioural assessment locomotion assay
Using DanioVision (Noldus Information Technologies, the Netherlands) locomotion of 6 dpf larvae was tracked. Each larva was placed individually in a well of a 48-well plate with 200 µL of E3 embryo medium. The study was conducted at a constant 28°C and 3000 lux. The protocol started with 30 min basal activity measurements, followed by a 5-min protocol in which taps in random order were generated in intervals varying between 2 and 35 s. Afterward, the complete larval body was used for sdhb genotyping. Larvae were pooled based on genotype and data grouped per phenotype were exported to Microsoft Excel (version 1906). The distance moved was used as read-outs for locomotion to detect possible differences between the three different genotypes.
Determination of cardiac output in embryonic zebrafish hearts
Embryos were treated with 1-phenyl-2-thiourea (PTU) at 20–24 h post-fertilization (hpf) to prevent pigmentation. At 6 dpf, embryos were anesthetized in 0.1% MS-222 and embedded in 0.3% agarose (UltraPure, Thermo Fisher Scientific) prepared in E3 medium containing 16 mg/mL MS-222. Imaging was performed at 28°C using a Leica DM IRBE inverted light microscope (Leica Microsystems) with a Hamamatsu C9300-221 high-speed CCD camera (Hamamatsu Photonics). Embryonic heart imaging was conducted at 150 frames per seconds (fps) using Hokawo 2.1 imaging software (Hamamatsu Photonics) for a duration of 10 s per embryo. Using ImageJ, heart rate was calculated. Then, volumes were analyzed using ImageJ by drawing a fit-to-ellipse on top of the ventricle at end-diastole and end-systole, to obtain the major and minor axes of the ellipse. Averages of three measurements per heart were determined. End diastolic and end systolic volume (EDV/ESV) were calculated by: (4/3) × (π) × (major axis/2) × ((minor axis/2)2). Stroke volume (SV) by: EDV−ESV. Cardiac output (CO) by: SV × heart rate.
Mitochondrial complex activity measurements
After genotyping, ten larval bodies (6 dpf) were pooled and snap-frozen in liquid N2 and stored at −80°C. Defrosted material was homogenized in 1450 μL 10mM Tris–HCl, pH 7.6. Then, 250 μL 1.5 M sucrose was added, followed by 10 min 14,000 g centrifugation at 2°C. Supernatant was discarded and the pellet resuspended in 200 μL 10mM 3Tris-HCl, pH 7.6. The enzymatic activities of complex 1–5 and citrate synthase and the sample protein content were assayed spectrophotometrically as previously described (Rodenburg 2011). Assays were performed in duplicate using a Konelab 20XT auto-analyzer (Thermo Fisher Scientific).
Mass spectrometry of Krebs cycle metabolites and amino acids
Using a mass spectrometry-based assay, metabolite involved in the TCA-cycle were measured in WT, heterozygous sdhb, and homozygous sdhb larvae. After genotyping, ten frozen larval bodies (6 dpf) were pooled. The larval samples were weighed, homogenized in 100 μL methanol together with an internal standard mixture, centrifuged to precipitate insolubles, and dried in a speed vac concentrator. Larval metabolite extracts were resuspended in mobile phase and analyzed by ultrahigh pressure liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) as described by Richter et al. (2019). Data were normalized based on weight.
Western blot
Samples for Western blot analyses were prepared from ten pooled snap-frozen total zebrafish larvae. Samples were denatured in Laemmli buffer containing 10 mM dithiothreitol and applied to sodium dodecyl sulfate-PAGE. Blots were incubated with primary antibody against SDHB (1:2000, 21A11AE7; Abcam) and horseradish peroxidase-conjugated secondary antibodies (1:10,000). Expression was corrected for β-actin (1:10.000, A5441, Sigma Aldrich) and normalized to the WT siblings.
Analysis of gene expression
Total RNA was isolated from 2 and 6 dpf larvae. Larvae were immediately frozen in liquid nitrogen, and total RNA per individual larvae was isolated using Trizol reagent according to the manufacturer’s instructions and treated with DNase-I. When appropriate, an additional ethanol precipitation step was performed to obtain RNA of high purity. Samples containing 300 ng RNA were used to prepare cDNA in duplicate using iScript (Bio-Rad) according to the manufacturer’s instructions.
Real-time quantitative PCR (RT-qPCR) and reverse transcriptase PCR were performed with SYBR Green Supermix (Bio-Rad). Primer oligonucleotides sequences used for amplification are provided in Supplementary Table 1 (see section on supplementary materials given at the end of the article). Before measurements, RT-qPCR primer pairs were validated by generation of standard curves with serially diluted cDNA to ensure the efficiencies of RT-qPCR, which were nearly 100%. The cycling conditions were as follows: 95°C for 3 min, 40 cycles of 95°C for 15 s, 60°C for 60 s, with a melting curve of 65°C–95°C, 5 se (ΔT +05°C/cycle). Expression levels are calculated relative to an index of two control amplicons, namely, eukaryotic elongation factor 1-alpha 1 (eef1a1/1), and ribosomal protein L13 (rpl13).
Immunohistochemistry
Larval zebrafish (6 dpf) from homozygous, heterozygous sdhb mutants and their WT siblings were cryoprotected with 10% sucrose in PBS for 30 min prior to embedding in OCT compound (Tissue-Tek, 4583, Sakura). After embedding, samples were slowly frozen in melting isopentane. To assess morphology, cryosections (7 μm thickness) were fixed for 10 min with paraformaldehyde (PFA; 4%, w/v), stained with hematoxylin and eosin and analyzed with a Zeiss Axioskop light microscope.
Transmission electron microscopy (TEM)
Larval zebrafish (6 dpf) were fixed at 4°C overnight in a freshly prepared mixture of 2.5% (v/v) glutaraldehyde and 2% PFA (w/v) in 0.1 M sodium cacodylate buffer (pH 7.4). After rinsing in buffer, specimens were post-fixated in a freshly prepared mixture, containing 1% osmium tetroxide and 1% potassium ferrocyanide in 0.1 M sodium cacodylate buffer (pH 7.4), at room temperature during 2 h. After rinsing, tissues were dehydrated through a graded series of ethanol and embedded in epon. Ultrathin (rostrocaudally) sections (70 nm) were collected on Formvar coated grids, subsequently stained with 2% uranyl acetate and Reynold’s lead citrate, and examined with a Jeol1010 electron microscope. Using Adobe Photoshop version 8.0, TEM images were adjusted for brightness and contrast. To compare the degree of vesiculation in the inner mitochondrial segments of the various experimental groups, quantitative TEM analysis was accomplished. To this end, 8000× magnification images of the central retina (50% middle arc length) were acquired.
Statistical analysis
Graphpad Prism software (Version 5.03 for Windows, Graphpad Software, www.graphpad.com) was used to generate scatter plots, calculate mean values, and perform statistical analyses. Log-rank (Mantel–Cox) test was used for the survival curve analysis, one-way ANOVA with Tukey’s post hoc test was used for the swim bladder quantification, metabolomics analyses, and Danio Vision quantification analysis. Kruskall–Wallis test with Dunn’s Multiple Comparison post hoc test was used for enzymatic activity analyses. The Student’s t-test was used for cardiac output analyses.
Results
Zebrafish sdhb, counterpart of human SDHB
Bioinformatic analysis found two sdhb paralogues in zebrafish and a high degree of sequence similarity (81% amino acid identity, 86% similarity) between zebrafish sdhb protein and human SDHB. The zebrafish sdhb gene is located on chromosome 22 at position 62,668–67,430 of the forward strand, consists of eight exons with a total length of 843 bases, and encodes a protein of 280 amino acids (Fig. 1). According to Interpro, the protein domains are conserved between zebrafish sdhb and human SDHB (Fig. 1). Both, the Bgee expression database and spatial RNA sequencing method of the Bakkers group provide evidence that sdhb is broadly expressed in zebrafish at several stages of development. Zebrafish sdhb contains no damaging amino acid substitutions when compared to human SDHB using Polyphen (Supplementary Fig. 1). Together, these data indicate that zebrafish sdhb is an expressed and functional ortholog of human SDHB.
Zebrafish have one functional paralogue of SDHB. Schematic presentation of human and zebrafish succinate dehydrogenase subunit B domain structure. The location of the mutation in sdhbrmc200 is schematically depicted. The two different protein domains, 2Fe-2S ferredoxin-type indicated by blue squatre and 4Fe-4S ferredoxin-type indicated by green oval, are conserved between zebrafish and human SDHB. Clustal W (1.81) protein sequence alignment of the one functional zebrafish protein sdhb paralogue is aligned with human SDHB, identifying functionally equivalent amino acids across the protein structure. Amino acid sequences are denoted in single-letter code. ‘*’ indicates that the residues in that column are identical in all sequences, ‘:’ indicates highly similar substitutions have been observed and ‘.’ indicates semi-similar substitutions are observed.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
Zebrafish have one functional paralogue of SDHB. Schematic presentation of human and zebrafish succinate dehydrogenase subunit B domain structure. The location of the mutation in sdhbrmc200 is schematically depicted. The two different protein domains, 2Fe-2S ferredoxin-type indicated by blue squatre and 4Fe-4S ferredoxin-type indicated by green oval, are conserved between zebrafish and human SDHB. Clustal W (1.81) protein sequence alignment of the one functional zebrafish protein sdhb paralogue is aligned with human SDHB, identifying functionally equivalent amino acids across the protein structure. Amino acid sequences are denoted in single-letter code. ‘*’ indicates that the residues in that column are identical in all sequences, ‘:’ indicates highly similar substitutions have been observed and ‘.’ indicates semi-similar substitutions are observed.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
Zebrafish have one functional paralogue of SDHB. Schematic presentation of human and zebrafish succinate dehydrogenase subunit B domain structure. The location of the mutation in sdhbrmc200 is schematically depicted. The two different protein domains, 2Fe-2S ferredoxin-type indicated by blue squatre and 4Fe-4S ferredoxin-type indicated by green oval, are conserved between zebrafish and human SDHB. Clustal W (1.81) protein sequence alignment of the one functional zebrafish protein sdhb paralogue is aligned with human SDHB, identifying functionally equivalent amino acids across the protein structure. Amino acid sequences are denoted in single-letter code. ‘*’ indicates that the residues in that column are identical in all sequences, ‘:’ indicates highly similar substitutions have been observed and ‘.’ indicates semi-similar substitutions are observed.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
The second (partially annotated) sequence is located on chromosome 23 at position 24,618,698 through 24,621,023 of the reverse strand. The 5’ and the 3’ end of this predicted gene are not annotated. In addition, so far no evidence was found for expression of this gene (Kruse et al. 2016). ClustalW amino acid alignment of the sequence with SDHB paralogs of other species and Polyphen predictions (Supplementary Figs 1 and 2) shows that zebrafish sdhb has substitutions of 72 of the 145 highly conserved amino acids, contain an extra stretch of sequence and misses two stretches of sequence compared to all other sequences. Moreover, Polyphen predicted that zebrafish sdhb contains 23 amino acid substitutions that are probably damaging and one substitution that is possibly damaging when these substitutions would occur in the human SDHB sequence (Supplementary Fig. 1). These data together indicate a non-functional SDHB paralog, most likely an eroded, untranscribed remnant of the fish-specific genome duplication.
Generation of the zebrafish sdhbrmc200 mutant using CRISPR/cas9
The first exon of sdhb was targeted using a single gRNA (targeting sequence 5’-GTTCACCGGCCCGCCGTCA-3’), which was injected together with Cas9 protein into eggs at the 1-cell stage. sdhbrmc200 contains a 13 bp frameshift mutation at the sdhb exon1-intron1 boundary (c. 57_63 + 64_69del; p.Val20ArgfsTer43) that is predicted to result in a premature termination of translation of succinate dehydrogenase iron-sulfur subunit B (Fig. 2A and B). On protein level, sdhb expression was decreased in 6-day-old homozygous sdhb larvae compared to WT siblings (Fig. 2C), sdhb expression was reduced by 50% in heterozygous sdhb larvae compared to WT siblings. In addition, RT-qPCR analysis showed a decrease in gene expression in homozygous sdhb larvae compared to heterozygous sdhb and WT siblings both at 2 dpf and 6 dpf (Fig. 2D).
Generation and validation of the sdhbrmc200 mutant. (A) The location of the mutation in sdhbrmc200 (c.57_63 + 64_69del; p.Val20ArgfsTer43) is depicted, a 13 bp frameshift mutation in sdhb exon1-intron1 boundary, predicted to yield a premature stop codon. (B) PCR analysis of sdhb genotype. Genomic DNA was extracted and used for PCR amplification. Expected amplicons are WT (123 bp), heterozygous sdhb (123 and 110 bp) and homozygous sdhb larvae (110 bp). (C) Western blot analysis of homozyous sdhb larvae. Protein expression of SDHB (~31 kDa) was measured in WT larvae and homozygous sdhb larvae. Ten larvae per condition were pooled. Protein expression was corrected for β-actin. (D) Gene expression analysis at 2 dpf and 6 dpf. RT-qPCR analysis between individual heterozygous sdhb (2 dpf n = 17; 6dpf n = 17), homozygous sdhb (2 dpf n = 13; 6 dpf n = 17) and WT larvae (2 dpf n = 11; 6 dpf n = 14) showed a significant reduction in heterozygous and homozygous sdhb larvae compared to WT siblings at both 2 and 6 dpf. One-way ANOVA with Tukey’s post hoc test, **P < 0.01 and ***P < 0.001.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
Generation and validation of the sdhbrmc200 mutant. (A) The location of the mutation in sdhbrmc200 (c.57_63 + 64_69del; p.Val20ArgfsTer43) is depicted, a 13 bp frameshift mutation in sdhb exon1-intron1 boundary, predicted to yield a premature stop codon. (B) PCR analysis of sdhb genotype. Genomic DNA was extracted and used for PCR amplification. Expected amplicons are WT (123 bp), heterozygous sdhb (123 and 110 bp) and homozygous sdhb larvae (110 bp). (C) Western blot analysis of homozyous sdhb larvae. Protein expression of SDHB (~31 kDa) was measured in WT larvae and homozygous sdhb larvae. Ten larvae per condition were pooled. Protein expression was corrected for β-actin. (D) Gene expression analysis at 2 dpf and 6 dpf. RT-qPCR analysis between individual heterozygous sdhb (2 dpf n = 17; 6dpf n = 17), homozygous sdhb (2 dpf n = 13; 6 dpf n = 17) and WT larvae (2 dpf n = 11; 6 dpf n = 14) showed a significant reduction in heterozygous and homozygous sdhb larvae compared to WT siblings at both 2 and 6 dpf. One-way ANOVA with Tukey’s post hoc test, **P < 0.01 and ***P < 0.001.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
Generation and validation of the sdhbrmc200 mutant. (A) The location of the mutation in sdhbrmc200 (c.57_63 + 64_69del; p.Val20ArgfsTer43) is depicted, a 13 bp frameshift mutation in sdhb exon1-intron1 boundary, predicted to yield a premature stop codon. (B) PCR analysis of sdhb genotype. Genomic DNA was extracted and used for PCR amplification. Expected amplicons are WT (123 bp), heterozygous sdhb (123 and 110 bp) and homozygous sdhb larvae (110 bp). (C) Western blot analysis of homozyous sdhb larvae. Protein expression of SDHB (~31 kDa) was measured in WT larvae and homozygous sdhb larvae. Ten larvae per condition were pooled. Protein expression was corrected for β-actin. (D) Gene expression analysis at 2 dpf and 6 dpf. RT-qPCR analysis between individual heterozygous sdhb (2 dpf n = 17; 6dpf n = 17), homozygous sdhb (2 dpf n = 13; 6 dpf n = 17) and WT larvae (2 dpf n = 11; 6 dpf n = 14) showed a significant reduction in heterozygous and homozygous sdhb larvae compared to WT siblings at both 2 and 6 dpf. One-way ANOVA with Tukey’s post hoc test, **P < 0.01 and ***P < 0.001.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
sdhbrmc200 mutants are viable, but shorter lived
After heterozygous sdhb mutants were raised to adulthood, homozygous sdhb mutants were generated from a heterozygous incross obeying Mendelian inheritance. Genotyping revealed no adult homozygous sdhb mutants and survival was examined during the first 15 days after fertilization (Fig. 3). Up to 6 dpf, survival of wild type, heterozygous and homozygous mutants was similar. After 6 dpf, homozygous sdhb mutants showed significantly enhanced mortality compared to both heterozygous and WT siblings (P < 0.001). By 14 dpf, all homozygous sdhb mutants had died. No differences in survival between heterozygous sdhb and WT siblings was observed (P = 0.83).
Survival in sdhb mutants. Survival rate (%) was significantly reduced in sdhbrmc200 homozygous mutants as compared to heterozygous sdhb and WT siblings (n = 71 homozygous sdhb larvae, n = 159 heterozygous sdhb larvae, and n = 83 WT larvae from two replicates). No significant differences were observed between sdhb heterozygous and WT siblings. Log-rank (Mantel–Cox) test, ***P < 0.001.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
Survival in sdhb mutants. Survival rate (%) was significantly reduced in sdhbrmc200 homozygous mutants as compared to heterozygous sdhb and WT siblings (n = 71 homozygous sdhb larvae, n = 159 heterozygous sdhb larvae, and n = 83 WT larvae from two replicates). No significant differences were observed between sdhb heterozygous and WT siblings. Log-rank (Mantel–Cox) test, ***P < 0.001.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
Survival in sdhb mutants. Survival rate (%) was significantly reduced in sdhbrmc200 homozygous mutants as compared to heterozygous sdhb and WT siblings (n = 71 homozygous sdhb larvae, n = 159 heterozygous sdhb larvae, and n = 83 WT larvae from two replicates). No significant differences were observed between sdhb heterozygous and WT siblings. Log-rank (Mantel–Cox) test, ***P < 0.001.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
Morphology
Comparative morphological analysis of heterozygous sdhb, homozygous sdhb and WT siblings (6 dpf) revealed either an absent or partially inflated swim bladder in the majority of heterozygous and homozygous sdhb larvae (Fig. 4A). Quantification of the swim bladder area showed 52,9% and 62,4% reduction in heterozygous and homozygous mutants compared to WT (Fig. 4B), for which the classification into the three types (0 = absent swim bladder inflation, 1 = partial swim bladder inflation and 2 = normal swim bladder inflation) is shown in Fig. 4C. Heart, liver and kidney appeared to be similar in sdhb mutants and WT siblings. No tumors were observed in 6-day-old sdhb mutant larvae. The intestinal lining showed shorter mucosal folds and an increased density of goblet cells indicating intestinal inflammation (Supplementary Fig. 3). Mitochondrial morphology and numbers did not differ at 6 dpf between homozygous sdhb larvae and WT siblings in brain and muscle tissue (Supplementary Fig. 4).
(A) Morphology of 6dpf zebrafish larvae of the three different genotypes. (B) Swim bladder area of complete inflation, partial or non-inflated swim bladder was quantified using ImageJ and plotted per genotype. The mean volume surface area taken by the swim bladder is significantly reduced between both WT and heterozygous sdhb larvae and WT and homozygous sdhb larvae (n = 41 homozygous sdhb larvae, n = 19 heterozygous sdhb larvae, and n = 53 WT larvae from 2 biological replicates). (C) Phenotypic quantification of absent swim bladder (class 0), partly inflated (class 1) and normal (class 2), and (n = 41 homozygous sdhb larvae, n = 19 heterozygous sdhb larvae, and n = 53 WT larvae from 2 biological replicates). One-way ANOVA with Tukey’s post hoc test, ***P < 0.001. Scale bar: 300 µm.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
(A) Morphology of 6dpf zebrafish larvae of the three different genotypes. (B) Swim bladder area of complete inflation, partial or non-inflated swim bladder was quantified using ImageJ and plotted per genotype. The mean volume surface area taken by the swim bladder is significantly reduced between both WT and heterozygous sdhb larvae and WT and homozygous sdhb larvae (n = 41 homozygous sdhb larvae, n = 19 heterozygous sdhb larvae, and n = 53 WT larvae from 2 biological replicates). (C) Phenotypic quantification of absent swim bladder (class 0), partly inflated (class 1) and normal (class 2), and (n = 41 homozygous sdhb larvae, n = 19 heterozygous sdhb larvae, and n = 53 WT larvae from 2 biological replicates). One-way ANOVA with Tukey’s post hoc test, ***P < 0.001. Scale bar: 300 µm.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
(A) Morphology of 6dpf zebrafish larvae of the three different genotypes. (B) Swim bladder area of complete inflation, partial or non-inflated swim bladder was quantified using ImageJ and plotted per genotype. The mean volume surface area taken by the swim bladder is significantly reduced between both WT and heterozygous sdhb larvae and WT and homozygous sdhb larvae (n = 41 homozygous sdhb larvae, n = 19 heterozygous sdhb larvae, and n = 53 WT larvae from 2 biological replicates). (C) Phenotypic quantification of absent swim bladder (class 0), partly inflated (class 1) and normal (class 2), and (n = 41 homozygous sdhb larvae, n = 19 heterozygous sdhb larvae, and n = 53 WT larvae from 2 biological replicates). One-way ANOVA with Tukey’s post hoc test, ***P < 0.001. Scale bar: 300 µm.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
Ambulatory activity
Upon visual inspection, obvious abnormalities in swimming behavior were observed in homozygous sdhb larvae, they showed impaired ambulatory activity. No differences in locomotor activity between heterozygotes and WT larvae were observed. During a light-dark locomotion test, 6 dpf larvae displayed elevated locomotor activity in the dark and decreased activity in the light as shown in Fig. 5. Homozygous sdhb mutants showed a lower basal activity during the first 10 min in the dark at baseline compared to their heterozygous and WT siblings (Fig. 5A and C). Homozygous sdhb mutants are able to respond to a light–dark transition; however, they have reduced endurance compared to heterozygous and WT siblings (Fig. 5A and B). No differences were observed between heterozygous sdhb mutants and WT siblings. Upon these results, cardiac performance was measured. Cardiac output was significantly decreased in homozygous sdhb larvae compared to WT siblings (Fig. 5D).
Locomotor activity of 6 dpf homozygous sdhb, heterozygous sdhb and WT siblings under alternating light-dark cycles and cardiac performance. (A) The average distance moved (mm/min) is plotted on the y-axis and time (minutes) plotted on the x-axis; black and white bars represent dark and light periods of 10 min, respectively. (B) Quantification mean distance moved of dark period 2, 3 and 4. The 10-min time period is divided into periods of 2 min. The homozygous sdhb larvae respond to the dark (min 3–4), however a faster decrease (min 7–8 and 9–10) in locomotor activity is observed compared to heterozygous and WT siblings. (C) Quantification of the first dark period. A lower basal activity is observed in homozygous sdhb larvae compared to heterozygous sdhb and WT siblings. (A, B and C) (n = 39 homozygous sdhb larvae, n = 59 heterozygous sdhb larvae, and n = 42 WT larvae from three replicates). One-way ANOVA with Tukey’s post hoc test , **P < 0.01, ***P < 0.001. (D) Cardiac performance in 6 dpf old larvae. A decrease in cardiac output (nl/minute) is observed in homozygous sdhb mutants (n = 20) compared to WT siblings (n = 21). Student’s t-test, **P < 0.01.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
Locomotor activity of 6 dpf homozygous sdhb, heterozygous sdhb and WT siblings under alternating light-dark cycles and cardiac performance. (A) The average distance moved (mm/min) is plotted on the y-axis and time (minutes) plotted on the x-axis; black and white bars represent dark and light periods of 10 min, respectively. (B) Quantification mean distance moved of dark period 2, 3 and 4. The 10-min time period is divided into periods of 2 min. The homozygous sdhb larvae respond to the dark (min 3–4), however a faster decrease (min 7–8 and 9–10) in locomotor activity is observed compared to heterozygous and WT siblings. (C) Quantification of the first dark period. A lower basal activity is observed in homozygous sdhb larvae compared to heterozygous sdhb and WT siblings. (A, B and C) (n = 39 homozygous sdhb larvae, n = 59 heterozygous sdhb larvae, and n = 42 WT larvae from three replicates). One-way ANOVA with Tukey’s post hoc test , **P < 0.01, ***P < 0.001. (D) Cardiac performance in 6 dpf old larvae. A decrease in cardiac output (nl/minute) is observed in homozygous sdhb mutants (n = 20) compared to WT siblings (n = 21). Student’s t-test, **P < 0.01.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
Locomotor activity of 6 dpf homozygous sdhb, heterozygous sdhb and WT siblings under alternating light-dark cycles and cardiac performance. (A) The average distance moved (mm/min) is plotted on the y-axis and time (minutes) plotted on the x-axis; black and white bars represent dark and light periods of 10 min, respectively. (B) Quantification mean distance moved of dark period 2, 3 and 4. The 10-min time period is divided into periods of 2 min. The homozygous sdhb larvae respond to the dark (min 3–4), however a faster decrease (min 7–8 and 9–10) in locomotor activity is observed compared to heterozygous and WT siblings. (C) Quantification of the first dark period. A lower basal activity is observed in homozygous sdhb larvae compared to heterozygous sdhb and WT siblings. (A, B and C) (n = 39 homozygous sdhb larvae, n = 59 heterozygous sdhb larvae, and n = 42 WT larvae from three replicates). One-way ANOVA with Tukey’s post hoc test , **P < 0.01, ***P < 0.001. (D) Cardiac performance in 6 dpf old larvae. A decrease in cardiac output (nl/minute) is observed in homozygous sdhb mutants (n = 20) compared to WT siblings (n = 21). Student’s t-test, **P < 0.01.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
Complex-ІІ deficiency and succinate accumulation in homozygous sdhb larvae
Spectrophotometric analyses of respiratory chain enzyme activities revealed an isolated deficiency of complex 2 in lysates of homozygous sdhb zebrafish (P < 0.05), with normal activities of complexes 1, 3, 4 and 5 (Fig. 6). Tissue metabolite profiling revealed an increase in succinate and lactate (P < 0.01), and a decrease in aspartate (P < 0.001) and asparagine (P < 0.05) levels in homozygous sdhb mutants compared to heterozygous and WT siblings (P < 0.001) compared to WT and heterozygous mutants. The heterozygous sdhb larvae showed no differences in metabolite levels compared to WT siblings.
Enzymatic activity and metabolomics of 6 dpf old larvae. (A) Enzymatic activity of Electron Transport Chain complexes 1–5 in lysates of 6 dpf zebrafish (n = 10 pooled per genotype, two replicates). The activity of complexes is expressed in IU per mg mitochondrial protein. Complex 2 activity is significantly decreased in homozygous sdhb larvae compared to WTs. (B) Abundance of metabolites compared between the three different genotypes (WT, heterozygous and homozygous sdhb larvae). Total lysates of 10 different larvae were pooled, measurements were performed in quadruplicate for WT larvae and quintuplicate for heterozygous and homozygous sdhb larvae. A significant increase of succinate and lactate is observed in homozygous sdhb larvae, and a significant decrease of aspartate, asparagine is observed in homozygous sdhb larvae compared to WT siblings. Kruskall Wallis test with Dunn’s Multiple Comparison post hoc test for enzymatic activity analysis and one-way ANOVA with Tukey’s post hoc test for metabolomics analysis, *P < 0.05, **P < 0.01, ***P < 0.001.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
Enzymatic activity and metabolomics of 6 dpf old larvae. (A) Enzymatic activity of Electron Transport Chain complexes 1–5 in lysates of 6 dpf zebrafish (n = 10 pooled per genotype, two replicates). The activity of complexes is expressed in IU per mg mitochondrial protein. Complex 2 activity is significantly decreased in homozygous sdhb larvae compared to WTs. (B) Abundance of metabolites compared between the three different genotypes (WT, heterozygous and homozygous sdhb larvae). Total lysates of 10 different larvae were pooled, measurements were performed in quadruplicate for WT larvae and quintuplicate for heterozygous and homozygous sdhb larvae. A significant increase of succinate and lactate is observed in homozygous sdhb larvae, and a significant decrease of aspartate, asparagine is observed in homozygous sdhb larvae compared to WT siblings. Kruskall Wallis test with Dunn’s Multiple Comparison post hoc test for enzymatic activity analysis and one-way ANOVA with Tukey’s post hoc test for metabolomics analysis, *P < 0.05, **P < 0.01, ***P < 0.001.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
Enzymatic activity and metabolomics of 6 dpf old larvae. (A) Enzymatic activity of Electron Transport Chain complexes 1–5 in lysates of 6 dpf zebrafish (n = 10 pooled per genotype, two replicates). The activity of complexes is expressed in IU per mg mitochondrial protein. Complex 2 activity is significantly decreased in homozygous sdhb larvae compared to WTs. (B) Abundance of metabolites compared between the three different genotypes (WT, heterozygous and homozygous sdhb larvae). Total lysates of 10 different larvae were pooled, measurements were performed in quadruplicate for WT larvae and quintuplicate for heterozygous and homozygous sdhb larvae. A significant increase of succinate and lactate is observed in homozygous sdhb larvae, and a significant decrease of aspartate, asparagine is observed in homozygous sdhb larvae compared to WT siblings. Kruskall Wallis test with Dunn’s Multiple Comparison post hoc test for enzymatic activity analysis and one-way ANOVA with Tukey’s post hoc test for metabolomics analysis, *P < 0.05, **P < 0.01, ***P < 0.001.
Citation: Endocrine-Related Cancer 28, 1; 10.1530/ERC-20-0308
Discussion
We successfully generated a sdhb mutant zebrafish model and demonstrated that homozygous sdhb knock-out in zebrafish leads to loss of complex 2 activity and elevated succinate levels, as has been shown for PPGLs and several different cell line models with SDH impairment (Pollard et al. 2005, Rao et al. 2013, Richter et al. 2014, Loriot et al. 2015, Lussey-Lepoutre et al. 2015). Functional analysis showed that homozygous sdhb larvae are viable, yet with a shorter lifespan. In the majority of the homozygous sdhb larvae incomplete inflation of the swim bladder was observed. Moreover, a lower basal swim activity and endurance was measured in the homozygous sdhb larvae. Here, we report the first viable sdhb animal model that reproduces important characteristics of SDHB mutated PPGL.
Interestingly, we did not find complete lack of complex 2 activity and a saw a faint band in western blots for homozygous knock-out fish, which may be explained by either partial rescue of the phenotype by maternal mRNA contribution from the yolk (Harvey et al. 2013, Despic & Neugebauer 2018) or alternative splicing (Smits et al. 2019). Decreased aspartate has also been described in SDHx mutated PPGL-tumors and in Sdhb-null cell lines indicating the lack of available carbons for aspartate synthesis from oxaloacetate (Richter et al. 2014, 2018, Lussey-Lepoutre et al. 2015). Elevations in lactate show the increased rate of glycolysis required by SDH-deficient cells to generate ATP and building blocks to sustain growth (Cervera et al. 2008, Lussey-Lepoutre et al. 2015, Kitazawa et al. 2017). Since the TCA-cycle is at least partly conserved from zebrafish to man, metabolic phenotypes observed in zebrafish can be related to the biology in human cells (Yang et al. 2018).
Additional morphological and behavioral analyses were performed to find the cause of death and specify the level of abnormalities in both heterozygous and homozygous sdhb mutants. First, we observed an obvious defect in swimbladder inflation. Larvae start to inflate their swimbladder at 3 dpf by ingesting small airbubbles, requiring access to the water-air interface to do so (Lindsey et al. 2010). Swimbladder inflation is a complex behavior requiring sensory, neural, and muscular integrity, and an incomplete swimbladder inflation could suggest a delay in development and/or difficulties in energy-consuming swimming behaviour. Both homozygous and heterozygous sdhb larvae showed a significant decrease in swimbladder filling. The majority of the homozygous sdhb mutants showed abberant swimming behaviour; the swimming behavior of heterozygous sdhb larvae was similar to that of WT siblings. Based on the lower basal activity and lowered endurance of homozygous sdhb mutant larvae shown by DanioVision analysis, we hypothesize that these observations results from defects in energy metabolism due to mitochondrial dysfunction and may therefore serve as a quick read-out to foretell phenotypes.
Indeed, the lifespan of homozygous sdhb larvae is reduced, but the larvae survive for almost 2 weeks whereas in other systemic knock-out animal models early embryonic lethality is observed. This offers a window of opportunity to study the model itself as well as interventions to rescue the phenotype aimed at the discovery of therapeutic strategies. Zebrafish larvae allow complex manipulation and study from fertilization onwards. All experiments described in this study are performed with larvae bred from heterozygous sdhb parents, since the homozygous sdhb mutants do not reach adulthood. Zebrafish embryos hatch externally to the mother and use maternal proteins in yolk to go through early developmental phases and this may explain why homozygous sdhb mutant zebrafish are viable, whereas Sdhb knock-out mice do not (Despic & Neugebauer 2018). In humans, bi-allelic mutations in SDHB result in Leigh-syndrome-like phenotype with largely impaired complex-II activity resulting in severe and progressive neurogeneration and myopathy with onset in infancy and poor prognosis (Alston et al. 2012, Gronborg et al. 2016). The lifespan of these patients usually varies between 1 and 5 years. This human phenotype of systemic mitochondrial failure in several ways resembles the phenotype of homozygous sdhb mutant larvae, including a decreased life span (Alston et al. 2012, Gronborg et al. 2016). The heterozygous larvae appear to have a normal lifespan, which corresponds with human heterozygous SDHB mutation carriers.
In patients with systemic bi-allelic sdhb mutations an increased white matter volume is observed (Alston et al. 2012, Gronborg et al. 2016). Ultrastructurally no major differences in mitochondria morphology was observed at day 6 in homozygous sdhb mutants comparable to the results in rat RS0 SDHB knock-out cells (Powers et al. 2020). To the best of our knowledge, electron microscopy data of only one patient with bi-allelic SDHB mutations are available; muscle histology of the vastus lateralis muscle at the age of 1 year demonstrated reduced histochemical activity of succinate dehydrogenase, and electron microscopy revealed sparse lipid droplets, some enlarged mitochondria with party disrupted cristae, and abundant glycogen in between myofibrils (Gronborg et al. 2016). Possibly, the damage in patients is visible at another timepoint than mentioned in the paper, since patients with bi-allelic sdhb mutations first show normal development until the age of 1 year, followed by sudden deterioration. Time kinetic analyses in zebrafish larvae could give valuable insights on when and where this transition might take place. Unexpectedly, intestinal inflammation as evidenced by shorter primary and secondary mucosal folds and increased number of goblet cells were observed. The role of succinate in intestinal inflammation has been described before in rats, mice, pigs, and in in vitro studies with human colon carcinoma cells (Connors et al. 2018). High succinate levels also cause mucosal erosion, submucosal edema, and inflammatory cell infiltration (Connors et al. 2018), similar to what we observed in our transgene fish. This finding emphasizes the broad role of succinate metabolism and the unique opportunity to study the effects of succinate levels in further detail in this fish model.
No tumor formation was recognized in sdhb knock-out fish so far. The homozygously mutated sdhb larvae show a severe phenotype and have a life span that is probably too short to allow the development of PPGL. This observation is in line with children with bi-allelic SDHB mutations, in whom to our knowledge no PPGLs were reported. In human carriers with a mono-allelic SDHB mutation, a second hit in the form of a somatic mutation in the unaffected SDHB allele is required for the development of a tumor. The disease penetrance among these carriers is incomplete, estimated at 42% at age 70 (Rijken et al. 2018). PPGLs are first diagnosed in SDHB mutation carriers in the early 30s (Olson et al. 2016). Specific triggers that are responsible for a second hit and subsequent tumor formation are unknown. So far, no tumor formation was observed in the adult heterozygous sdhb mutant zebrafish. It could be that this species is less prone to spontaneously occurring somatic mutations. An alternative option to acquire a tumor model would be to apply gene-editing, generating a tissue-specific mutant, crossed with the currently obtained systemic mutant, to create a homozygous mutation in chromaffin cells specifically. Such an approach is feasible in zebrafish using modern tools, and is part of our ongoing studies. Additional strategies that previously have been used in the search for a Sdhb rodent tumor model could also be applied in our model, including conditional inactivation, incross high calorie diet, radiation, drugs and hormones (Lepoutre-Lussey et al. 2016, Powers et al. 2020). Although short-lived, the zebrafish model opens a window of opportunity to test toxicity of certain drugs which could be validated in the available Sdhb rodent tumor models.
In addition, the current model could give new and valuable insights into the signaling pathways involved in, and activated upon, succinate accumulation that eventually could lead to tumorigenesis. In the pertinent literature several hypotheses have been proposed to explain PPGL tumorigenesis: ROS accumulation, altered DNA and histon methylation profiles, increased HIF levels, and decreased DNA repair by homologous recombination defects are all hypothesized to lead to tumorigenesis driven by high succinate levels. Although the exact dynamics and involvement of these pathways is unknown (Cascon et al. 2019). The current model gives us the opportunity to study the consequences of sdhb inactivation and could give us more insight in the first steps of disease development, specifically the role and consequences of high succinate accumulation. Further, transgenic zebrafish have a great potential to build (semi-)high throughput drug screening assays to identify the contributions of different pathways to the disease phenotype (Macrae & Peterson 2015).
In summary, we identified for the first time an in vivo sdhb model with a diseased phenotype mimicking human SDHB-associated PPGL tumor characteristics and isolated complex-II deficiency. This mutant is therefore vital to further our insight in pathophysiological mechanisms of these diseases and possibly opens doors to develop new therapeutic approaches.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/ERC-20-0308.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the Paradifference Foundation.
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