dldhcri3 zebrafish exhibit altered mitochondrial ultrastructure, morphology, and dysfunction partially rescued by probucol or thiamine
Manuela Lavorato, Donna Iadarola, Cristina Remes, Prabhjot Kaur, Chynna Broxton, Neal D. Mathew, Rui Xiao, Christoph Seiler, Eiko Nakamaru-Ogiso, Vernon E. Anderson, Marni J. Falk
Manuela Lavorato, Donna Iadarola, Cristina Remes, Prabhjot Kaur, Chynna Broxton, Neal D. Mathew, Rui Xiao, Christoph Seiler, Eiko Nakamaru-Ogiso, Vernon E. Anderson, Marni J. Falk
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Research Article Genetics Metabolism

dldhcri3 zebrafish exhibit altered mitochondrial ultrastructure, morphology, and dysfunction partially rescued by probucol or thiamine

Abstract

Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disease caused by variants in DLD, the E3 subunit of mitochondrial α-keto (or 2-oxo) acid dehydrogenase complexes. DLD disease symptoms are multisystemic, variably manifesting as Leigh syndrome, neurodevelopmental disability, seizures, cardiomyopathy, liver disease, fatigue, and lactic acidemia. While most DLD disease symptoms are attributed to dysfunction of the pyruvate dehydrogenase complex, the effects of other α-keto acid dehydrogenase deficiencies remain unclear. Current therapies for DLD deficiency are ineffective, with no vertebrate animal model available for preclinical study. We created a viable Danio rerio (zebrafish) KO model of DLD deficiency, dldhcri3. Detailed phenotypic characterization revealed shortened larval survival, uninflated swim bladder, hepatomegaly and fatty liver, and reduced swim activity. These animals displayed increased pyruvate and lactate levels, with severe disruption of branched-chain amino acid catabolism manifest as increased valine, leucine, isoleucine, α-ketoisovalerate, and α-ketoglutarate levels. Evaluation of mitochondrial ultrastructure revealed gross enlargement, severe cristae disruption, and reduction in matrix electron density in liver, intestines, and muscle. Therapeutic modeling of candidate therapies demonstrated that probucol or thiamine improved larval swim activity. Overall, this vertebrate model demonstrated characteristic phenotypic and metabolic alterations of DLD disease, offering a robust platform to screen and characterize candidate therapies.

Authors

Manuela Lavorato, Donna Iadarola, Cristina Remes, Prabhjot Kaur, Chynna Broxton, Neal D. Mathew, Rui Xiao, Christoph Seiler, Eiko Nakamaru-Ogiso, Vernon E. Anderson, Marni J. Falk

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Figure 4

Decreased survival, abnormal gross morphology, and reduced swimming activity in dldh–/– zebrafish larvae.

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Decreased survival, abnormal gross morphology, and reduced swimming acti...
(A) dldh–/– larvae had reduced survival relative to WT larvae, with mortality starting at 8 dpf; n = 300 each strain, across 3 biological replicates; log-rank (Mantel-Cox) test: P < 0.0001; median survival of dldh–/–: 10 dpf. Death was defined by absent heartbeat. (B) Morphological analysis at 7 dpf showed dldh–/– zebrafish larvae had enlarged liver (white arrowhead), darker intestine (black arrows) with universal finding of uninflated swim bladder (asterisk in WT), relative to WT larvae. Scale bar: 0.5 mm. (C) Larval swimming activity (ZebraBox platform and ZebraLab software) tracing showed reduced dldh–/– mutant swimming (open circles) in dark periods (gray) at 7 dpf. Larvae were exposed to 3 consecutive 10-minute 100% light (yellow) and 10-minute dark cycles following a light/dark acclimation period. Each point shows average (mean + SEM) activity of 84 larvae/strain. (D) dldh–/– swimming activity at 7 dpf during dark cycles analyzed by AUC, with group comparison. ****P < 0.0001 by unpaired Student’s t test. Data are shown as mean ± SEM. n = 84 larvae across > 3 biological replicate experiments per strain.