Opa1 Is Required for Proper Mitochondrial Metabolism in Early Development

Citation: Rahn JJ, Stackley KD, Chan SSL ( Opa1 Is Required for Proper Mitochondrial Metabolism in Early Development Jennifer J. Rahn 0 Krista D. Stackley 0 Sherine S. L. Chan 0 Mark R. Cookson, National Institutes of Health, United States of America 0 Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina , Charleston, South Carolina , United States of America Opa1 catalyzes fusion of inner mitochondrial membranes and formation of the cristae. OPA1 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, pgc1a, 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. - Funding: This work was supported by National Institutes of Health awards R00ES015555, 5P20RR024485-02 and 8 P20 GM103542-02, startup funds provided by the Medical University of South Carolina, and the South Carolina Clinical and Translational Research Institute/Medical University of South Carolina CTSA, National Institutes of Health/National Center for Research Resources (UL1RR029882 and UL1 TR000062). Imaging facilities for this research were supported, in part, by Cancer Center Support Grant P30 CA138313 to the Hollings Cancer Center, Medical University of South Carolina. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Mitochondria are dynamic organelles, and undergo fission, fusion and replication/biogenesis in response to energy demands and stress [1,2]. OPA1 encodes a dynamin-related GTPase targeted to the inner mitochondrial membrane and has been demonstrated to play critical roles in mitochondrial fusion, cristae remodeling, and sequestration and release of cytochrome c [3]. In humans, the OPA1 protein exists as 69 isoforms that are generated by alternate splicing between exons 4, 4b, and 5b and/ or proteolytic processing [4]. Some OPA1 isoforms are differentially located within the mitochondrial intermembrane space, and the various isoforms may facilitate division of labor for the many roles OPA1 plays in the cell [4]. In vitro studies have demonstrated the importance of OPA1 in mitochondrial form and function, as down-regulation of OPA1 leads to disruption of inner mitochondrial membrane fusion in addition to impaired respiration (bioenergetics) and loss of mitochondrial membrane potential [5,6]. Additionally, OPA1 has a role in maintaining mitochondrial DNA (mtDNA) stability and integrity by impacting genome mixing that occurs during mitochondrial fusion [7]. Mutations in OPA1 are associated with autosomal dominant optic atrophy (ADOA) in humans, a disease characterized by progressive loss of visual acuity, desensitization of central visual field, optic nerve pallor, and eventual blindness [8,9]. Histologically, advanced stages of the disease are characterized by selective loss of the retinal ganglion cell (RGC) layer and ascending atrophy of the optic nerve [9]. To date, over 200 pathogenic mutations have been identified in OPA1 ranging in location throughout the coding sequence with the exception of exons 4, 4b and 5, and include substitutions, deletions, and insertions [10]. Haploinsufficiency appears to play a major role in pathogenicity of ADOA suggesting that homozygous mutations may be embryonic lethal [10]. As with other diseases associated with mutations in mitochondrial genes, disease severity and age of onset appear to vary even within family members bearing the same mutation [11]. Up to 20% of patients bearing OPA1 mutations develop additional phenotypes including deafness, progressive external ophthalmoplegia, myopathy, and neuromuscular complications. This more severe set of phenotypes is often referred to as OPA1-plus [12], and is variably associated with mtDNA mutations and deletions as well as mtDNA depletion [13] as is often noted in patients with other mitochondrial diseases [11,13,14]. Two heterozygous mouse models of OPA1 have been developed to explore the disease characteristics of ADOA. One model introduces a premature stop codon at Q285 in exon 8 [15], while the other contains a splice site mutation in intron 10 resulting in the skipping of exon 10 (329355 aa) [16]. Heterozygous mice of both genotypes exhibit a 50% reduction in OPA1 transcript levels in the retinal tissue along with a similar reduction in OPA1 protein in a variety of other tissues. Interestingly, these models appear to recapitulate the slow visual degeneration but do not display loss of RGCs seen in human patients [17]. Despite these mouse models, questions still remain as to how depletion of the ubiquitously expressed OPA1 protein results in defects in mitochondrial function and why this depletion results in the apparent tissue specific phenotype. Furthermore, OPA1 null mice and homozygotes lose viability at embryonic day (E) 9, which may explain the lack of identified patients with homozygous OPA1 mutations [17]. The specific defects that occur before E9 resulting in mortality in these mice have not been characterized. 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 [18]. Zebrafish can produce hundreds of embryos in a single breeding and the embryos are transparent and develop outside of the mother, allowing for non-invasive observations of organ development [19]. Opa1 is ubiquitously expressed in zebrafish [20] as a single-copy gene and is 78% identical and 87% similar to the most abundant human OPA1 isoform (OPA1-4) at the protein level. In order to investigate the role of Opa1 in embryonic development, we developed a zebrafish model of Opa1 depletion and determined the functional consequences of Opa1 mediated mitochondrial dysfunction. Changes in Opa1 protein during zebrafish embryoni (...truncated)