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 3

Unchanged RC enzyme activity and mitochondrial respiration capacity in dldh–/– zebrafish larvae.

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Unchanged RC enzyme activity and mitochondrial respiration capacity in d...
(A) Measurement of the DLD (E3) activity in dldh–/– zebrafish showed significantly decreased activity as compared with WT; n = 3; ***P < 0.001 by Student’s t test. (B) Enzyme activities of complex I (CI), CII, and CIV and citrate synthase (CS); n = 3. Data are shown as mean ± SD. No significant differences between 7 dpf dldh/ larvae and WT by Student’s t test. (C) Oxygen flux measurements obtained by high-resolution polarography with an Oxygraph-2k (Oroboros) on 5 dpf zebrafish homogenate. Basal respiration is the measurement before addition of substrates. OXPHOSCI is the capacity of CI taken after the addition of glutamate. OXPHOSCI+CII is the capacity of both CI+CII taken after the addition of succinate. LEAKCI+CII is the nonphosphorylating electron transfer across the mitochondrial inner membrane. ETSCII is in the presence of succinate and rotenone. TMPD-Az is the ascorbate driven complex IV activity. A typical time course and experimental details are presented in Supplemental Figure 3C. None of the differences reached statistical significance by Welch’s t test. Data are shown as mean ± SD (n = 3).