Opa1 Is Required for Proper Mitochondrial Metabolism in Early Development

<div><p>Opa1 catalyzes fusion of inner mitochondrial membranes and formation of the cristae. <i>OPA1</i> mutations in humans lead to autosomal dominant optic atrophy. OPA1 knockout mice lose viability around embryonic day 9 from unknown reasons, indicating that OPA1 is essential for embryonic development. Zebrafish are an attractive model for studying vertebrate development and have been used for many years to describe developmental events that are difficult or impractical to view in mammalian models. In this study, Opa1 was successfully depleted in zebrafish embryos using antisense morpholinos, which resulted in disrupted mitochondrial morphology. Phenotypically, these embryos exhibited abnormal blood circulation and heart defects, as well as small eyes and small pectoral fin buds. Additionally, startle response was reduced and locomotor activity was impaired. Furthermore, Opa1 depletion caused bioenergetic defects, without impairing mitochondrial efficiency. In response to mitochondrial dysfunction, a transient upregulation of the master regulator of mitochondrial biogenesis, <i>pgc1a</i>, was observed. These results not only reveal a new Opa1-associated phenotype in a vertebrate model system, but also further elucidates the absolute requirement of Opa1 for successful vertebrate development.</p> </div

Primers for XL-PCR for mtDNA deletion assay.

<p>Primers for XL-PCR for mtDNA deletion assay.</p

Primers for RT-PCR.

<p>Primers for RT-PCR.</p

Western blot analysis.

<p><b>A.</b> Representative Western blot for Opa1 yolk-less protein in MMC and Opa1 morphants. Opa1 protein is reduced at 24, 48, and 72 hpf. Differences were isoform specific. Samples from 24 hpf were imaged from a separate blot. Contrast was adjusted to improve visualization. *indicates isoforms selected for densitometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059218#pone-0059218-g001" target="_blank">Figure 1B</a>). Western blot results have been replicated with at least four independent injections <b>B.</b> Quantification of most intense Opa1 isoforms identified by Western blot. All values were first normalized to β-actin protein levels.</p

Antisense morpholino sequences.

<p>Antisense morpholino sequences.</p

Phenotypic analyses of MMC (A, C, E, G, I, K, M, O) and TB (B, D, F, H, J, L, N, P) morphant embryos and larvae.

<p>Opa1 morphants at 24 hpf (<b>B</b>) have increased density or ‘graininess’ in the brain region (arrow) and smaller eyes. At 48 hpf, Opa1 morphants have hindbrain ventricle enlargements (arrow) and smaller eyes (<b>D</b>). Opa1 morphants at 48 hpf also have impaired circulation compared with MMC morphants and often has blood accumulation below the heart (arrow) (<b>F</b>). At 72 hpf, Opa1 morphants have larger yolk cells, smaller eyes, smaller hearts, small pectoral fin buds (<b>H</b>) and pericardial edema (<b>J</b>). Many Opa1 morphants had unlooped hearts (<b>L</b>). (<b>N</b>) is the same image as (<b>L</b>) with the heart margins outlined (solid line) and the midline indicated by a dashed line. By 96 hpf, the edema is still present and can involve the eyes (<b>P</b>).</p

Gene expression changes of mitochondrial morphology genes in MMC morphants (black) and Opa1 morphants (grey) normalized to MMC morphant levels.

<p>Significant increases in gene expression of mitochondrial fusion proteins (<b>A</b>) <i>opa1</i> (a, p = 0.003), (<b>B</b>) <i>mfn1</i> (b, p = 0.001) and (<b>C</b>) <i>mfn2</i> (c, p = 0.01) were observed in Opa1 morphants compared to MMC morphants. No differences were observed for (<b>D</b>) <i>drp1</i>, a mitochondrial fission protein. Error bars are shown +/− SEM, n = 5. P-values obtained by ANOVA.</p

Eye area and heart rate analyses for MMC (black) and Opa1 morphants (grey).

<p><b>A.</b> Eye area was measured by tracing the circumference of individual eyes using AxioVision software. N = 9–12. *p-value <0.05, **p-value <0.01 by Student's 2-tailed t-test. <b>B.</b> Heart rates were measured by counting beats per min for individuals injected with MMC or TB morpholino. N = 34 (48 hpf), n = 44 (72 hpf). *p-value <0.01 by Student's 2-tailed t-test.</p

Gene expression changes in MMC morphants (black) and Opa1 morphants (grey) normalized to MMC morphant levels.

<p>Significant increases in gene expression <i>pgc1a</i> (a, p = 0.02) and <i>peo1</i> (b, p = 0.002) were observed in <i>opa1</i> morphants compared to MMC morphants. Error bars are shown +/- SEM, n = 5. P-values obtained by ANOVA.</p

Partitioning of total basal respiration changes with zebrafish embryonic development.

<p>(<b>A</b>) The components of total respiration, per embryo. Non-mitochondrial respiration (dark grey), mitochondrial respiration (black), respiration due to ATP turnover (white) and respiration due to proton leak (light grey). Mean +/− SEM; n = 8. (<b>B</b>) Total basal respiration (100%) is composed of a non-mitochondrial fraction (white) and a mitochondrial fraction. The mitochondrial fraction is further composed of the respiration associated with proton leak (black) and ATP turnover (grey). The proportion of total respiration due to non-mitochondrial respiration was greatest at 3 hpf (p<0.0001) (a) and 7 hpf (p<0.02) (b) as determined by one-way ANOVA and Student-Newman-Keuls post hoc test. Non-mitochondrial respiration as a proportion of total respiration did not change between 12 and 48 hpf (p>0.05). At 3 hpf, the proportion of total respiration due to mitochondrial ATP turnover was lowest when compared with 7, 30 and 48 hpf (p<0.05) (c). Mitochondrial ATP turnover did not change much at other time-points. Proton leak as a proportion of total respiration at 3 hpf and 7 hpf was not significantly different to zero as determined by Student's t-test (p>0.05). Proton leak was significantly higher at 12 hpf (d) and 24 hpf (e) than at 3 and 7 hpf, as determined by one-way ANOVA and Student-Newman-Keul's post hoc test (p>0.05). Furthermore, proton leak was significantly higher at 24 hpf as compared to 48 hpf (p<0.05) (e).</p