Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Apr 20;15(4):281.
doi: 10.1038/s41419-024-06622-9.

Zebrafish polg2 knock-out recapitulates human POLG-disorders; implications for drug treatment

Raquel Brañas Casas  1 , Alessandro Zuppardo  2 , Giovanni Risato  1   3 , Alberto Dinarello  1   4 , Rudy Celeghin  3 , Camilla Fontana  1   5 , Eleonora Grelloni  1 , Alexandru Ionut Gilea  6 , Carlo Viscomi  2 , Andrea Rasola  2 , Luisa Dalla Valle  1 , Tiziana Lodi  6 , Enrico Baruffini  6 , Nicola Facchinello  7 , Francesco Argenton  8 , Natascia Tiso  9
Affiliations

Zebrafish polg2 knock-out recapitulates human POLG-disorders; implications for drug treatment

Raquel Brañas Casas et al. Cell Death Dis. .

Abstract

The human mitochondrial DNA polymerase gamma is a holoenzyme, involved in mitochondrial DNA (mtDNA) replication and maintenance, composed of a catalytic subunit (POLG) and a dimeric accessory subunit (POLG2) conferring processivity. Mutations in POLG or POLG2 cause POLG-related diseases in humans, leading to a subset of Mendelian-inherited mitochondrial disorders characterized by mtDNA depletion (MDD) or accumulation of multiple deletions, presenting multi-organ defects and often leading to premature death at a young age. Considering the paucity of POLG2 models, we have generated a stable zebrafish polg2 mutant line (polg2ia304) by CRISPR/Cas9 technology, carrying a 10-nucleotide deletion with frameshift mutation and premature stop codon. Zebrafish polg2 homozygous mutants present slower development and decreased viability compared to wild type siblings, dying before the juvenile stage. Mutants display a set of POLG-related phenotypes comparable to the symptoms of human patients affected by POLG-related diseases, including remarkable MDD, altered mitochondrial network and dynamics, and reduced mitochondrial respiration. Histological analyses detected morphological alterations in high-energy demanding tissues, along with a significant disorganization of skeletal muscle fibres. Consistent with the last finding, locomotor assays highlighted a decreased larval motility. Of note, treatment with the Clofilium tosylate drug, previously shown to be effective in POLG models, could partially rescue MDD in Polg2 mutant animals. Altogether, our results point at zebrafish as an effective model to study the etiopathology of human POLG-related disorders linked to POLG2, and a suitable platform to screen the efficacy of POLG-directed drugs in POLG2-associated forms.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. polg2ia304 line generation, survival and developmental delay.
A Schematic representation of zebrafish polg2 and its editing by CRISPR/Cas9. B polg2ia304/ia304 individuals fail to survive to 4 weeks post fertilization. Data were analysed by χ2 test; p = 0.4165; p = 0.9802; p = 0.5617; p = 0.0438; p = 0.0226; p < 0.0001; 3 dpf (n = 120), 6 dpf (n = 115), 12 dpf (n = 89), 20 dpf (n = 70), 25 dpf (n = 50), 30 dpf (n = 46). Gene expression analysis performed to assess polg2 (C) and polg (D) mRNA levels in polg2ia304 mutant line. Values are reported as mean ± SEM of three independent experiments and analysed by Games-Howell’s multiple comparison test (C) and ordinary one-way ANOVA followed by Tukey’s test (D); *p < 0.05; **p < 0.005; ***p < 0.001; polg2+/+ (n = 6 × 10-larva pools), polg2+/ia304 (n = 6 × 10-larva pools), polg2ia304/ia304 (n = 4 × 10-larva pools). E Representative images of zebrafish polg2+/+ control and polg2ia304/ia304 mutant at 20 dpf, and polg2+/+ control and a polg2+/ia304 heterozygote at 90 dpf. Scale bar: 2 mm (top); scale bar: 1 cm (bottom). F Body length analysis in wt (polg2+/+), heterozygous (polg2+/ia304) and homozygous (polg2ia304/ia304) individuals. Values are expressed as mean ± SEM. Statistics were assessed by Mann-Whitney test; ****p < 0.0001; polg2+/+ 6 dpf (n = 25), polg2+/ia304 6 dpf (n = 41), polg2ia304/ia304 6 dpf (n = 22); polg2+/+ 20 dpf (n = 39), polg2+/ia304 20 dpf (n = 74), polg2ia304/ia304 20 dpf (n = 45); polg2+/+ 90 dpf (n = 8), polg2+/ia304 90 dpf (n = 7).
Fig. 2
Fig. 2. polg2ia304 behavioural defects assessed by locomotor analysis.
A, C, E Effect of light stimuli on locomotion in polg2ia304/ia304 mutants at 6, 15 and 20 dpf, respectively. White fields indicate light exposition time, while grey fields represent dark periods. The analysis assessed the average total distance swum at 6 dpf (B), 15 dpf (D) and 20 dpf (F). Values derived from 3 independent biological replicates and are reported as mean ± SEM. Data were analysed using ordinary one-way ANOVA and Tukey’s test (B) or Kruskal-Wallis together with Dunn’s multiple comparison test (D, F); *p < 0.05; **p < 0.005; ***p < 0.001; for 6 dpf polg2+/+ (n = 19), polg2+/ia304 (n = 33), polg2ia304/ia304 (n = 13); for 15 dpf polg2+/+ (n = 32), polg2+/ia304 (n = 58), polg2ia304/ia304 (n = 45); for 20 dpf polg2+/+ (n = 57), polg2+/ia304 (n = 131), polg2ia304/ia304 (n = 49). G Effect of tapping stimulus on locomotion in wt, heterozygous and polg2ia304/ia304 individuals at 8 dpf. Tapping stimuli were delivered in a 20-s time window with an interstimulus interval of 1 s. H Average response to maximum tapping stimuli given as the total distance covered by polg2ia304/ia304 homozygotes during tap stress assay, compared to wt and heterozygous sibs. Results are expressed as mean ± SEM. For statistical analysis, the Kruskal-Wallis test corrected with Dunn’s multiple comparison test was applied; *p < 0.05; polg2+/+ (n = 38), polg2+/ia304 (n = 72), polg2ia304/ia304 (n = 26), from 3 independent experiments.
Fig. 3
Fig. 3. Effects of polg2 mutation on high energy demanding tissues.
A, C Comparison of cardiac tissues at 20 dpf, reporting differences in atrial (a) and ventricular (v) dimensions and an altered trabecular network (t) in mutants. Scale bar: 5 mm. B Histological sections of polg2+/+ and polg2ia304/ia304 skeletal muscle tissue at 20 dpf, displaying disorganized fibres and altered somite boundaries in mutants. Arrows: detachment of skeletal muscle fibres. Scale bar: 5 mm. D TEM analysis of 20 dpf skeletal muscle of wt (polg2+/+) and homozygous mutants (polg2ia304/ia304), the latter displaying altered organization of myofibrils. Scale bar: 1 µm. E TEM analysis of mitochondria in 20 dpf heart of wt (polg2+/+) and homozygous mutants (polg2ia304/ia304). Scale bar: 500 nm. TEM analysis of 20 dpf skeletal muscle from wt (polg2+/+) (F) and homozygous mutants (polg2ia304/ia304) (G) confirms altered morphology of myofibrils and mitochondrial cristae (black arrowheads) in mutants. Scale bar: 500 nm. H Quantification of inter-cristae distance (in nm) in the cardiac region. Data are shown as mean ± SEM and statistics were done using unpaired Student’s t-test. I Quantification of heart mitochondrial (mt) aberrant morphologies. For statistical analysis, the χ2 test was applied; N = 8 independent TEM images per condition, in triplicate. J Quantification of inter-cristae distance (in nm) in the skeletal muscle. Values are reported as mean ± SEM. Statistics were assessed using unpaired Student’s t-test; ****p < 0.0001. K Quantification of skeletal muscle mt aberrant morphologies. χ2 test was used for statistical analysis, N = 8 independent TEM images per condition, in triplicate. ****p < 0.0001. L Number of wide inter-cristae areas, considered when at least equal to 100 × 100 nm, counted for each organelle in the skeletal muscle. Data are shown as mean ± SEM. Mann-Whitney test was used for statistics; ****p < 0.0001.
Fig. 4
Fig. 4. Defects in mitochondrial content, morphology and metabolism under polg2 KO.
A Confocal images of the Tg(Hsa.Cox8a:MLS-EGFP)ia301 mitochondrial marker (mito:EGFP) in polg2+/+, polg2+/ia304 and polg2ia304/ia304 zebrafish at 6 dpf. Scale bar: 10 µm. B Relative quantification of the mito:EGFP transgene at 6 dpf. Data are reported as mean ± SEM. Statistics were assessed using the Kruskal-Wallis test followed by Dunn’s multiple comparison test; *p < 0.05; ****p < 0.0001; n = 10 independent larvae per condition. Comparison of relative abundances of the mitochondrial nd1 (C) and nd2 (D) genes in wt (polg2+/+), heterozygous (polg2+/ia304) and homozygous (polg2ia304/ia304) zebrafish at 6 dpf. Values are shown as mean ± SEM. Ordinary one-way ANOVA (C) or Games-Howell’s multiple comparison test (D) were applied for statistics; **p < 0.005; ***p < 0.001; C polg2+/+ (n = 6 × 10-larva pools), polg2+/ia304 (n = 6 × 10-larva pools), polg2ia304/ia304 (n = 6 × 10-larva pools); (D) polg2+/+ (n = 5 × 10-larva pools), polg2+/ia304 (n = 8 × 10-larva pools), polg2ia304/ia304 (n = 5 × 10-larva pools) from 3 independent biological replicates. E Analysis of the nd1 gene at 20 dpf. Values are reported as mean ± SEM. For statistical analysis, the Kruskal-Wallis test corrected with Dunn’s multiple comparison test was used; **p < 0.005; polg2+/+ (n = 6), polg2+/ia304 (n = 7), polg2ia304/ia304 (n = 7) from 3 independent experiments. F Diagram depicting the oxygen consumption rate (OCR) profile by Seahorse assay in 4 dpf wt (polg2+/+), heterozygous (polg2+/ia304) and homozygous (polg2ia304/ia304) larvae under basal conditions, FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone)-induced maximal respiratory capacity stimulation and ROT/AA (Rotenone/Antimycin A)-mediated inhibition. Two-way ANOVA followed by Tukey’s multiple comparison test was applied for statistical significance; *p < 0.05 between polg2+/+ and polg2ia304/ia304 individuals. G Quantification of basal OCR in 4 dpf zebrafish. Data are shown as mean ± SEM and were analysed by ordinary one-way ANOVA; *p < 0.05; polg2+/+ (n = 9), polg2+/ia304 (n = 24), polg2ia304/ia304 (n = 8) from 3 independent replicates. H Imaging of HRE:EGFP (Hif-hypoxia) reporter at 6 dpf: an example of wt (above, left), heterozygous (above, right) and homozygous (below) mutant larva. Scale bar: 1 mm. I Integrated density analysis of Hif-hypoxia signalling activation in 6-dpf wt (polg2+/+), heterozygous (polg2+/ia304) and homozygous (polg2ia304/ia304) larvae. Values are expressed as mean ± SEM and analysed by ordinary one-way ANOVA; **p < 0.005; polg2+/+ (n = 6), polg2+/ia304 (n = 26), polg2+/ia304 (n = 52), polg2ia304/ia304 (n = 21), coming from 3 independent experiments.
Fig. 5
Fig. 5. Analysis of CLO rescuing effects under polg2 KO.
A mtDNA content in treated (+) and non-treated (-) polg2+/+, polg2+/ia304 and polg2ia304/ia304 individuals, exposed to 3 μM CLO from 2 dpf to 6 dpf; Data are shown as mean ± SEM, each dot corresponds to a 10-fish pool. Statistics were assessed using ordinary one-way ANOVA, followed by Tukey’s multiple comparison test; *p < 0.05; **p < 0.005; ****p < 0.0001; polg2+/+ (n = 6 ×10-larva pools), polg2+/+ + CLO (n = 5 x 10-larva pools), polg2+/ia304 (n = 10 x 10-larva pools), polg2+/ia304 + CLO (n = 10 x 10-larva pools), polg2ia304/ia304 (n = 7 x 10-larva pools), polg2ia304/ia304 + CLO (n = 6 ×10-larva pools), from 3 independent biological replicates. B Effect of light stimuli on locomotion in treated (+) and non-treated (-) polg2+/+, polg2+/ia304 and polg2ia304/ia304 individuals, exposed to 2 μM CLO from 10 dpf to 15 dpf. White fields indicate light exposition time, while grey fields represent dark periods. Two-way ANOVA followed by Dunn’s multiple comparison test was applied for statistical significance; polg2+/+ (n = 34), polg2+/+ + CLO (n = 32), polg2+/ia304 (n = 70), polg2+/ia304 + CLO (n = 50), polg2ia304/ia304 (n = 35), polg2ia304/ia304 + CLO (n = 26). C Maximum response to light stimuli, expressed in mm (distance swum) among the three genotypes (polg2+/+, polg2+/ia304 and polg2ia304/ia304) in treated (+) and non-treated (-) fish. Values are reported as mean ± SEM. Statistical analysis was done by Kruskal-Wallis test followed by Dunn’s multiple comparison test; **p < 0.005; *p < 0.05; polg2+/+ (n = 34), polg2+/+ + CLO (n = 32), polg2+/ia304 (n = 70), polg2+/ia304 + CLO (n = 50), polg2ia304/ia304 (n = 35), polg2ia304/ia304 + CLO (n = 26), from 3 independent replicates and in triplicate.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Nogueira C, Almeida LS, Nesti C, Pezzini I, Videira A, Vilarinho L, et al. Syndromes associated with mitochondrial DNA depletion. Ital J Pediatr. 2014;40:34. doi: 10.1186/1824-7288-40-34. - DOI - PMC - PubMed
    1. El-Hattab AW, Scaglia F. Mitochondrial DNA depletion syndromes: review and updates of genetic basis, manifestations, and therapeutic options. Neurotherapeutics. 2013;10:186–98. doi: 10.1007/s13311-013-0177-6. - DOI - PMC - PubMed
    1. El-Hattab AW, Craigen WJ, Scaglia F. Mitochondrial DNA maintenance defects. Biochim Biophys Acta Mol Basis Dis. 2017;1863:1539–55. doi: 10.1016/j.bbadis.2017.02.017. - DOI - PubMed
    1. Basel D. Mitochondrial DNA depletion syndromes. Clin Perinatol. 2020;47:123–41. doi: 10.1016/j.clp.2019.10.008. - DOI - PubMed
    1. Viscomi C, Zeviani M. MtDNA‐maintenance defects: syndromes and genes. J Inher Metab Dis. 2017;40:587–99. doi: 10.1007/s10545-017-0027-5. - DOI - PMC - PubMed
-