Facchinello et al., 2021 - Efficient clofilium tosylate-mediated rescue of POLG-related disease phenotypes in zebrafish. Cell Death & Disease   12:100 Full text @ Cell Death Dis.

Fig. 1 po<italic>lg</italic> morphants characterization and specific signalling pathway alteration.

A, B Molecular validation of polg-specific morpholino oligos. Semi-quantitative RT-PCR from total cDNA of not injected (not inj), mismatched-morpholino (mismMO) injected controls and polg-specific morphants (MO), at 1 and 2 dpf, detects-specific alteration of polg transcripts. Arrowheads indicate altered cDNA products exclusively in polg morphant embryos (A). Control actb1 transcripts appear unaltered under all conditions (B). M marker. PCR experiments were performed in triplicate on cDNA samples derived from N = 30 whole embryos per condition. Quantifications are displayed in the (M) chart. C, D Cardiac dilation in polg morphants. Frontal views of control (C) and polg morphant (D) embryos at 2 dpf. Note the enlarged atrium in the morphant; a atrium, v ventricle. Atrial measurements are displayed in the (N) chart. E, F TEM analysis of mitochondria in the skeletal muscle of 2-dpf embryos: normal mitochondrial morphology in controls (E); fused and swollen mitochondria in morphants (altered areas indicated by black arrowheads (F). GL Analysis of Hif-Hypoxia and Wnt signalling pathways under polg deficiency. Bright field (G, I, J) imaging shows a decreased body length, in the absence of overt dysmorphology, in 1 and 2-dpf morphant embryos; measurements are displayed in the (O) chart. Fluorescence (H, K, L) imaging of Hif-Hypoxia (HRE Hif-Hypoxia Responsive Elements) and Wnt signalling reporters shows strong activation of the Hif-Hypoxia pathway under polg-deficieny, in both green (H) and red (K, L) transgenic background. On the contrary, Wnt signalling appears unchanged (compare K with L). Relative intensity (R.I.) quantifications of the fluorescent signals are displayed in the (P) and (Q) charts. Statistical tests: one-way ANOVA followed by Tukey’s test; ***p < 0.0001; **p < 0.001; n.s. not significant; N = 6 samples per condition, in triplicate.

Fig. 2 Editing of the zebrafish <italic>polg</italic> gene.

A Zebrafish polg exons are shown as color-coded boxes corresponding to protein domains of the Polg peptide. Black arrows show the position of the CRISPR/Cas9-targeted site in exon 2 of the polgia302 mutant line, and the missense mutation in exon 22 of the polgsa9574 mutant line. In the polg+/+ (up) vs. polg−/− (down) sequences, the CRISPR/Cas9-targeted site is in italic and the protospacer adjacent motif (PAM) is underlined. The new stop codons generated in both mutants are indicated with * = TGA, in red. B, Cpolgia302/ia302 individuals fail to survive to three weeks (B), while part of polgsa9574/9574 animals can survive to adulthood (C). In the Kaplan–Meier survival curve for polgsa9574, time is shown in days post-fertilization (dpf); N = 50 individuals per genotype. The Log Rank test was used for statistical analysis; polg+/+ vs. polgsa9574/sa9574: p < 0.0001. D Body length analysis in wt (polg+/+), heterozygous (polg+/ia302) and homozygous (polgia302/ia302) individuals. The body length of zebrafish polgia302/ia302 mutants is significantly reduced compared to controls at 15 dpf. Heterozygotes body length is indistinguishable from wt at all considered stages. N = 8, in triplicate; **p < 0.01. E Body length analysis in wt (polg+/+), heterozygous (polg+/sa9574) and homozygous (polgsa9574/sa9574) individuals. The body length of zebrafish polgsa9574/sa9574 mutants is significantly reduced compared to control and heterozygous individuals at all considered stages. N = 8, in triplicate; *p < 0.05; **p < 0.01. F Representative images of a zebrafish polg+/+ control and a polgia302/ia302 mutant at 16 dpf; a polg+/+ control is compared with a polg+/ia302 heterozygote at 90 dpf. G Representative images of a zebrafish polg+/+ control and a polgsa9574/sa9574 mutant at 16 and 90 dpf.

Fig. 3 Effects of Polg mutation on heart and gonads of adult zebrafish.

A Cardiac EE histology shows a clear reduction of the thickness of the trabecular network in mutant hearts; a atrium, cm compact myocardium, t trabeculae, v ventricle. B Ovarian EE histology detects oocyte maturation defects in mutant females; black arrowheads indicate debris of atretic follicles. po primary oocyte, cao cortical-alveolar oocyte, vo vitellogenic oocyte. C Testicular EE histology detects a reduced amount of spermatozoa (sp) in mutant males. Concentration (D), velocity (E) and viability (F) of ejaculated sperm. Depicted is mean ± SEM. ***p < 0.001; N = 10, in triplicate. G Illustrative image of fluorescence staining evidencing a higher amount of dead (red) over alive (green) sperm in polgsa9574/sa9574 males.

Fig. 4 Effects of Polg mutation on liver, skeletal muscle and mitochondria.

AD Representative Z projections of 3D confocal images of wt and polgsa9574/sa9574 liver at 8 and 16 dpf. The quantification of liver-specific fluorescence from the lfabp10a:DsRed transgene (E) indicates reduced liver size in 16 dpf mutants. FI Reference bright field images (F, G) and light microscopy analysis of muscle birefringence (H, I) in wt and polgsa9574/sa9574 embryos at 3 dpf. The birefringence quantification (see Methods) shows reduced signal in mutants (J). KN Confocal images of the Tg(Hsa.Cox8a:MLS-EGFP)ia301 mitochondrial marker (mitoEGFP) in wt (K, M) and polgsa9574/sa9574 (L, N) at 16 dpf. The relative quantification of integrated density (O) indicates a reduced mitochondrial mass. P The relative quantification of mtDNA copy number, in wt and polg sa9574 mutants, shows a significant reduction of mtDNA in homozygotes. Data are presented as mean ± SEM and were generated from three different experiments, each containing six embryos per genotype and treatment condition (*p < 0.05; **p < 0.01; N = 6, in triplicate). Q, R TEM analysis of mitochondria in 16 dpf skeletal muscle fibres (sm) of wt and polgsa9574/sa9574 detects vesicular cristae with “honeycomb” arrangement inside the mutated mitochondria (indicated by black arrowheads). S Quantification of aberrant morphologies; ***p < 0.001; N = 8 independent TEM images per condition, in triplicate.

Fig. 5 Altered signalling and mitochondrial dysfunction in <italic>polg</italic> mutants.

A The CREB signalling-targeted gene fosab is upregulated in 4 dpf polgsa9574/sa9574 mutants, compared to controls. B The Hif-Hypoxia signalling-targeted gene hbbe3 is upregulated in 4 dpf polgsa9574/sa95 mutants, compared to controls. CE Hif-Hypoxia signalling increases also in 6-dpf polgia302/ia302 mutants, as shown by the Hif-Hypoxia-responsive transgene HRE:mCherry. E Chart reporting the HRE fluorescence relative intensity (R.I.). F Diagram depicting the oxygen consumption rate (OCR) profile throughout Seahorse assay in 4 dpf wt and polgsa9574/sa9574 embryos under basal conditions, FCCP (p-triFluoromethoxyCarbonylCyanide Phenylhydrazone)-induced maximal respiratory capacity stimulation and ROT/AA (Rotenone/Antimycin A)-mediated inhibition. Three independent experiments were performed (N = 20). G Quantification of basal respiration in wt and polgsa9574/sa9574 mutants (N = 20, in triplicate).

Fig. 6 The CLO compound can rescue mitochondrial and cardio-skeletal defects in <italic>polg</italic> mutants.

A, B Comparison of relative abundances of the mitochondrial nd1 gene in the presence of CLO, in wt and polgsa9574/sa9574 zebrafish, untreated (A) or treated (B) with Ethidium Bromide (EtBr). polg+/+ zebrafish recover from EtBr-induced mtDNA depletion while polgsa9574/sa9574 mutants remain depleted of mtDNA. (A’, B’) Data in A’ and B’ charts were generated from three different experiments, each containing a pool of six embryos per genotype and treatment condition (N = 6, in triplicate). C Complex I activity in polgsa9574/sa9574 adult muscle tissue is significantly lowered, and can be rescued after CLO treatment. Data were generated from three different experiments, each containing a pool of four samples per genotype and treatment condition (N = 4, in triplicate). DF Retrograde signalling decrease after CLO treatment, based on the expression of the Hif-Hypoxia targets hbbe3 and pfkfb3 (D, E), and the CREB-responsive gene fosab (F); N = 5. G Muscle birefringence, reduced in polgsa9574/sa9574 mutants compared to siblings, can be rescued by CLO treatment. G’ Relative intensity (R.I.) quantifications of experiments shown in G; N = 10 per genotype. H The mitochondrial mass, analysed at 6 dpf by mitochondria-targeted EGFP Tg(Hsa.Cox8a:MLS-EGFP) in the skeletal muscle of all polgsa9574 genotypes, is reduced in homozygotes (compare C with A, B), and can be rescued by CLO treatment (DF). H’ Relative intensity (R.I.) quantifications of experiments shown in H; N = 9 per genotype. I The atrial diameter (dashed white lines), enlarged in 2-dpf polg morphants (C) compared to not injected (A) and control-injected (B) embryos, can be restored to normal values after CLO treatment (DF). Cardiac chambers are visualized by Tg(tg:EGFP,myl7:EGFP)ia300. I’ Quantifications of experiments shown in I; the atrial diameter is expressed in arbitrary units (A.U.); N = 6 per condition. J The heart rate, increased in polg morphants (MO) compared to controls (N.i.; mMO), can be restored to normal values after CLO treatment; N = 10 per condition; Bpm beats per minute. All charts in Fig. 6 display data as mean ± SEM; n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Acknowledgments:
ZFIN wishes to thank the journal Cell Death & Disease for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Cell Death Dis.