Drosophila melanogaster come modello di malattie mitocondriali umane: analisi funzionale dei geni deEthe1 e dSurf1

Ethylmalonic Encephalopathy (EE) is a rare autosomal recessive metabolic disorder caused by mutations in Ethe1, a nuclear gene encoding a mitochondrial matrix thioesterase. Although the function of ETHE1 protein is still unknown, the severe consequences of its malfunctioning indicate an important role of the Ethe1 gene product in mitochondrial homeostasis and energy metabolism (Tiranti et al., 2004). In order to understand the molecular pathogenesis underlying EE, it would be useful to establish a Drosophila melanogaster model in which the expression of CG30022 (dEthe1), the Drosophila Ethe1 homolog, is disrupted or impaired. I have thus set up transgenic Drosophila melanogaster lines in which it is possible to induce the post-transcriptional silencing of dEthe1 by double-stranded RNA interference. I was unable to detect any functional differences between knockdown and control flies. This lack of effects can be due to incomplete knockdown of dEthe1 mRNA, that mimicks a heterozygosity condition, asymptomatic in humans as well. In order to produce a complete null allele of dEthe1, we applied a gene targeting strategy that provides a way to knock-out genes in Drosophila using the organism's endogenous machinery of DNA repair and recombination. Since CG30022 is a small gene (1492 pb), we used the "two-step ends-in targeting" system (Xie & Golic, 2004) that allows precise deletions even in small target loci. Moreover, in Tilling, traditional chemical mutagenesis is followed by high-throughput screening for mutations in a target gene (Till et al., 2003). By this strategy, seven Drosophila lines carrying missense and frameshift mutations in dEthe1 gene were identified and characterized. On the basis of a dETHE1 three dimensional model, it was possible to predict the functional consequences of the amino acid changes characterizing each mutant. Moreover, we have characterized dEthe1 expression, both at a transcriptional and translational level, in Drosophila lines 0017965 and 104009 carrying a transposable element in the regulatory region and in the first exon of the dEthe1 gene, respectively. Leigh Syndrome (LS) is a progressive mitochondrial encephalopathy. The single most prevalent cause of LS are mutations in the Surf1 gene, which encodes a protein located in the inner membrane of mitochondria which is probably involved in cytochrome c oxidase assembly (Tiranti et al., 1999). Recently, a Drosophila melanogaster model for Leigh Syndrome has been characterized in which double-stranded RNA interference is used to induce the post-transcriptional silencing of CG9943 (dSurf1) gene, the D. melanogaster Surf1 homolog (Zordan et al., 2006). The structural and functional abnormalities at level of nervous system, as well as the global developmental arrest, observed in Drosophila knockdown flies are largely concordant with the human Leigh syndrome phenotype. Here, we have designed a method to assess if the phenotypic changes induced by double-stranded RNA interference are due to silencing of the targeted gene or to off-target effects. This method uses trans-complementation by a synthetic gene (dSurf1-synonymous) that encodes the same protein as the native gene but uses a different nucleotide sequence to escape RNAi-induced silencing (Kumar et al., 2006). We have generated transgenic lines in which it is possible to induce, by the UAS-GAL4 system, the simultaneous activation of dSurf1-synonymous expression and dsRNAi silencing of the endogenous Surf1 gene. This activation, induced via heat shock during early development, produces "rescued" flies, even if in a proportion that is lower than expected. The functional characterization of "rescued" flies shows a complete reversion of behavioral and electrophysiological abnormalities detectable in knockdown flies.