Efeitos da expressão da oxidase alternativa em mutantes da helicase mitocondrial de Drosophila melanogaster
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Data
2017-07-25
Autores
Rodrigues, Ana Paula Campos [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Mutações no gene nuclear humano C10orf2, que codifica a helicase mitocondrial Twinkle, são apontadas como causa da forma autossômica dominante da doença Oftalmoplegia Progressiva Externa (adPEO), associada a múltiplas deleções no DNA mitocondrial (DNAmt), e de outras doenças humanas. Duas mutações na Twinkle análogas àquelas encontradas em pacientes humanos (W441C e A442P) e uma mutação no sítio ativo (K388A) foram modeladas em Drosophila melanogaster, levando a defeitos na replicação do DNA mitocondrial, danos no processo de fosforilação oxidativa (OXPHOS) e desordens no desenvolvimento. A expressão transgênica da oxidase alternativa mitocondrial (AOX) tem se mostrado eficiente no combate a disfunções mitocondriais causadas por defeitos genéticos, principalmente por contornar os complexos III e IV da cadeia respiratória (CR). Assim, o objetivo deste projeto foi testar se a expressão da AOX poderia melhorar os efeitos deletérios causados por mutações na Twinkle no desenvolvimento de D. melanogaster. Dados fenotípicos mostraram que a expressão da AOX nos mutantes K388A e A422P da Twinkle foi incapaz de prevenir o fenótipo letal ainda em estágio larval ou pupal e também não alterou a depleção do DNAmt nessas moscas. Os mutantes W441C, na presença ou ausência da AOX, não tiveram problemas no desenvolvimento e nem mudanças no número de cópias de DNAmt. As linhagens superexpressando Twinkle tipo selvagem (WT) e AOX tiveram viabilidade normal e um aumento no número de cópias de DNAmt em adultos, não atribuído à presença da AOX. Ao medir a atividade de OXPHOS em tecidos não relacionados à metamorfose, prejuízos associados à expressão das Twinkles mutantes ou WT não foram detectados, nem alteração significativa pela presença da AOX. Portanto, nossos dados evidenciam Portanto, nossos dados evidenciam que a utilização da AOX como estratégia de terapia gênica para remediar casos de mutações na Twinkle apresenta certas limitações e que talvez o papel da helicase mitocondrial na fisiologia animal seja bem mais complexo do que mostrado até então.
Mutations in the human nuclear gene C10orf2, which encodes the mitochondrial replicative helicase Twinkle, are associated with cases of autosomal dominant Progressive External Ophthalmoplegia (adPEO) with multiple deletions in the mitochondrial DNA (mtDNA), and other diseases in humans. Two mutations analogous to those found in human patients (W441C and A442P) and an active site mutation (K388A) were modeled in Drosophila melanogaster. The expression of the mutant enzymes led to defects in mtDNA replication, impairment of mitochondrial oxidative phosphorylation (OXPHOS), and developmental disorders in flies. Transgenic expression of the mitochondrial alternative oxidase (AOX) has been shown to be efficient in combatting mitochondrial dysfunctions caused by genetic defects, mainly due to the bypass of complex III and IV. Thus, our aim was to test whether AOX expression can rescue the deleterious developmental effects caused by Twinkle mutations in D. melanogaster. Phenotypic data has shown that AOX expression in Twinkle mutants K388A and A422P was unable to prevent the lethal phenotype at the larval or pupal stage, and AOX was not effective against the mtDNA depletion of these flies. The W441C mutants, in the presence or absence of AOX, did not show developmental problems, nor changes in mtDNA copy number. Lines overexpressing the wild-type Twinkle (WT) and AOX had normal viability, in addition to an increase in mtDNA copy number in adults, not attributed to AOX. By measuring OXPHOS activity in tissues unrelated to metamorphosis, we detected no dysfunctions associated with the expression of mutant or WT Twinkle, nor a significant alteration by the presence of AOX. Our data indicates that the use of AOX as a gene therapy strategy to help cases of Twinkle mutations has limitations, and that perhaps the role of the mitochondrial replicative helicase in animal physiology is much more complex than previously thought.
Mutations in the human nuclear gene C10orf2, which encodes the mitochondrial replicative helicase Twinkle, are associated with cases of autosomal dominant Progressive External Ophthalmoplegia (adPEO) with multiple deletions in the mitochondrial DNA (mtDNA), and other diseases in humans. Two mutations analogous to those found in human patients (W441C and A442P) and an active site mutation (K388A) were modeled in Drosophila melanogaster. The expression of the mutant enzymes led to defects in mtDNA replication, impairment of mitochondrial oxidative phosphorylation (OXPHOS), and developmental disorders in flies. Transgenic expression of the mitochondrial alternative oxidase (AOX) has been shown to be efficient in combatting mitochondrial dysfunctions caused by genetic defects, mainly due to the bypass of complex III and IV. Thus, our aim was to test whether AOX expression can rescue the deleterious developmental effects caused by Twinkle mutations in D. melanogaster. Phenotypic data has shown that AOX expression in Twinkle mutants K388A and A422P was unable to prevent the lethal phenotype at the larval or pupal stage, and AOX was not effective against the mtDNA depletion of these flies. The W441C mutants, in the presence or absence of AOX, did not show developmental problems, nor changes in mtDNA copy number. Lines overexpressing the wild-type Twinkle (WT) and AOX had normal viability, in addition to an increase in mtDNA copy number in adults, not attributed to AOX. By measuring OXPHOS activity in tissues unrelated to metamorphosis, we detected no dysfunctions associated with the expression of mutant or WT Twinkle, nor a significant alteration by the presence of AOX. Our data indicates that the use of AOX as a gene therapy strategy to help cases of Twinkle mutations has limitations, and that perhaps the role of the mitochondrial replicative helicase in animal physiology is much more complex than previously thought.
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Palavras-chave
Twinkle, AOX, Disfunções mitocondriais, adPEO, Drosophila melanogaster, Mitochondrial disorders