Generation and characterization of a new model of OPA1-linked Dominant Optic Atrophy

Autor: Montagna, Aldo
Jazyk: angličtina
Rok vydání: 2017
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Popis: Mitochondria are very dynamic organelles with a crucial role in life and death of eukaryotic cells. These organelles regulate cellular energy generation, calcium and redox homeostasis, and apoptosis. To perform the cellular functions effectively, mitochondria continuously change their structure and morphology through protein machineries controlling fission and fusion process (mitochondrial dynamics). Strong evidence has emerged to implicate disturbed mitochondrial fusion and fission as central pathological components underpinning a number of childhood and adult-onset neurodegenerative disorders. Several proteins that regulate the morphology of the mitochondrial network have been identified, the most widely studied of which are Optic Atrophy 1 (OPA1), Mitofusin1 and 2 (Mfn1 and 2) and Dynamin Related Protein 1 (DRP1). OPA1 is a ubiquitously expressed dynamin-like GTPase in the inner mitochondrial membrane. It plays important roles in mitochondrial fusion, apoptosis, reactive oxygen species (ROS) and ATP production. Mutations of OPA1 result in autosomal Dominant Optic Atrophy (DOA), a common hereditary optic neuropathy characterized by retinal ganglion cell degeneration leading to optic neuropathy, symmetrical central visual loss and dyschromatopsia. The majority of OPA1 mutations result in premature termination codons, and the resultant truncated mRNA species are highly unstable, being rapidly degraded by protective surveillance mechanisms operating via nonsense-mediated mRNA decay. Haploinsufficiency, therefore, is a major disease mechanism in DOA, and the pathological consequences of a dramatic reduction in OPA1 protein levels is highlighted by those rare families who are heterozygous for microdeletions spanning the entire OPA1 coding region. Progressive visual failure remains the defining feature of DOA but, with greater availability of genetic testing, a specific OPA1 mutation in exon 14 (c.1334G>A, p.Arg445His) has been found to cause sensorineural deafness, ataxia, myopathy, peripheral neuropathy, and classical chronic progressive external ophthalmoplegia. This syndrome is called DOA plus. The molecular mechanisms linking OPA1 mutations and DOA are not fully understood. In this work a new model of OPA1-linked Dominant Optic Atrophy was generated in Drosophila melanogaster, in order to use it for studying DOA pathogenesis. The Drosophila OPA1 gene (dOPA1) shares 51.2% similarity with its human orthologue and the alignment of protein sequence of hOPA1 with dOPA1 shows that the domains most subjected to pathogenic mutations are well conserved. To address the pathophysiological mechanism of OPA1-linked DOA, we generated two dOPA1 mutants: OPA1 R417H, a mutant that carries in endogenous dOPA1 the mutation corresponding to R445H in humans; OPA1null carrying a microdeletion leading to production of a inactive truncated protein of 482 amino acids. To model these mutations we have used in vivo CRISPR/Cas9, a genome engineering system that has revolutionized genetic analysis in many organisms. For use in genome engineering the system requires two essential components: gRNA and Cas9-endonuclease. The gRNA recognizes a 20-nt target sequence next to a trinucleotide NGG protospacer adjacent motif (PAM) to direct Cas9-dependent cleavage of both DNA strands within the target sequence. Several groups have used the CRISPR/Cas9 system to induce targeted mutations in Drosophila, but differ in their approach to supply the Cas9 protein and gRNA components of the system. It has been demonstrated that two targeting gRNAs can be used to generated a large defined deletions and the Cas9 catalyzed gene replacement by homologous recombination. The experimental design of my work requires the following steps: generation of the gRNAs responsible for precisely targeting the genomic region where recombination should take place; generation of the dsDNA templates containing the desired genomic modifications to be introduced and homology arms for accurate recombination; choice of a screening method. The gRNAs guide the cut of the Cas9 on the genomic region of the dOPA1 gene through the target sequences and the PAM sites; the cut of genomic DNA favors homologous recombination with the dOPA1 mutated fragment cloned into dsDNA plasmid donor. The exogenous dOPA1 mutated gene in addition to the pathological mutations carries a silent mutation that introduces a novel BamHI restriction site necessary for screening the occurrence of homologous recombination events by restriction digest. The gRNAs and the dsDNA plasmids donors were microinjected in the embryo of the Drosophila line expressing Cas9 protein in the ovary under control of vasa regulatory sequences. The mutants were screened for the presence of the mutation through PCR on genomic DNA, restriction digest and sequencing of the OPA1 mutated fragment. After having verified that the mutants had mutations of interest without any other alterations, we described the phenotypic effects observed in these mutants. We also analyzed the mitochondria in the nervous and muscular systems using confocal microscopy and the mitochondrial functions through biochemical assays. Observations of the adults within the lines shows that both mutations in heterozygosity do not cause any evident morphological alterations. However, both mutations in homozygosity turned out to be lethal but differently R417H homozygous mutants develop until the second instar larva stage whereas the OPA1null homozygotes die earlier at the first instar larva stage. We were more interested in studying the heterozygote dOPA1 mutants because DOA is a dominant disease. The lifespan reduction of both dOPA1 mutants indicate that the heterozygous mutations of OPA1 is likely to cause systemic consequences probably affecting multiple processes. Since OPA1 is involved in mitochondrial dynamics we performed a series of experiments to analyze mitochondrial morphology in the neuronal and muscular systems of both dOPA1 mutants. Heterozygous dOPA1 mutants display defects of mitochondria morphology in nerves and muscles in Drosophila third instar larvae, mitochondria network shape is characterized by mild fragmentation and clusterization. Mitochondria function was analyzed on homozygous and heterozygous dOPA1 mutants. Mitochondrial respiration and the redox activity of respiratory complexes was decreased in both mutants. Furthermore heterozygous OPA1 R417H displayed more severe effects in some assays than OPA1null heterozygotes. This suggested that R417H mutation could interfere with the activity of the wild type copy of dOPA1 resulting in more severe phenotypes than those caused by the presence of a single loss of function allele. In conclusion, we have produced a model to study the Dominant Optic Atrophy which can be helpful to understand the pathogenesis of this disease caused by different classes of mutations within the OPA1 gene.
Databáze: OpenAIRE
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