Loss of sdhb in zebrafish larvae recapitulates human paraganglioma characteristics

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 behaviour were observed in homozygous sdhb mutantlarvae. 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 paediatric mitochondrial disease caused by loss of the SDHB gene.

Targeting the Redox Balance Pathway Using Ascorbic Acid in sdhb Zebrafish Mutant Larvae

“…As previously reported, homozygous sdhb larvae have a shortened lifespan (maximal two weeks of age) compared to heterozygous sdhb and wild-type siblings [22]. Additionally, these homozygous sdhb larvae display key metabolic characteristics of SDHB-associated PPGLs such as impaired mitochondrial complex II function and vastly increased succinate levels [22].…”

“…The development of novel therapeutic targets for metastatic SDHB-associated PPGLs is hampered by the limited availability of suitable in vivo models. In this study, we investigated the effects of Vitamin C in the sdhb rmc200 zebrafish model and thereby tested the suitability for drug screening experiments in this validated PPGL cancer model [22]. First, we revealed an imbalance in cellular redox homeostasis in homozygous sdhb zebrafish larvae by increased levels of ROS at baseline.…”

“…We previously identified that homozygous sdhb mutant larvae possess a lower basal activity and reduced endurance compared to heterozygous and wild-type siblings [22]. We designed a short protocol (<5 min) with randomized tapping stimuli to induce a robust startle response.…”

“…Ethical approval of the experiments was granted by Radboud University's Institutional Animal Care and Use Committee (IACUC, application numbers RU-DEC 2015-0098 and RU-DEC 2020-0030). Wild-type adult Oregon AB* zebrafish (Danio Rerio) and heterozygous adult sdhb rmc200 mutants were used [22]. Eggs were obtained from natural spawning.…”

“…Recently, we generated and characterised a systemic sdhb rmc200 knockout zebrafish model that mimics the metabolic properties of SDHB-associated PPGLs [22]. Homozygous sdhb rmc200 mutant larvae display a decreased lifespan due to decreased mitochondrial complex II activity and significant succinate accumulation, and they mimic important genomic and metabolic effects observed in SDHB-associated PPGL tumours [22]. In addition, a decreased mobility attributed to energy deficiency is observed.…”