Metabolic adaptation to mitochondrial dysfunction in D. melanogaster tko25t mutant model - transcriptional response and functional studies of the induced genes
ALA-HONGISTO, HANNA (2012)
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ALA-HONGISTO, HANNA
2012
Biokemia - Biochemistry
Biolääketieteellisen teknologian yksikkö - Institute of Biomedical Technology
Hyväksymispäivämäärä
2012-08-27Tiivistelmä
Background and Aims: Mitochondrial diseases, generally regarded as disorders affecting the OXPHOS system, manifest in a variety of clinical phenotypes.D. melanogaster tko25taffords a model for OXPHOS deficiency, resulting from a decreased intramitochondrial translational capacity, and has phenotypic characteristics similar to those presented in many human mitochondrial diseases. Thetko25tmodel exhibits systematic changes in the expression of several genes associated with metabolism, stress response and reproduction, in response to OXPHOS deficiency. A set of the most highly up-regulated key metabolic stress-response genes, namelyCG11659,CG17192,Fbp1,Fbp2,Hsp22andImpL3, was chosen for analysis. The aims of the present study were to test nutritional effects on expression up-regulation and to determine, if down-regulation of these genes by RNAi has a specific influence on development or physiological functions oftko25tflies. Thus, the present study aimed at understanding natural metabolic adaptation to mitochondrial dysfunction.
Methods: Nutritional effects on expression up-regulation were tested on four different culture media, with variable sugar and protein content, and measured using Q-RT-PCR. Drug-inducible GeneSwitch (GAL4-UAS) expression system and transgenicD. melanogasterRNAi lines were used for knockdown. The phenotypic effects of expression down-regulation were determined by performing developmental time and bang-sensitivity experiments. Bothtko25tand wild-type flies were studied.
Results: All the genes tested were up-regulated intko25ton all diets and in both sexes, compared to wild-type flies. In general, there were no major effects of diet, and the transcriptional response was quantitatively similar on all diets. With few exceptions, slightly increased expression levels were observed in wild-type flies grown on low-sugar diets. Functional RNAi lines were not found forCG11659orHsp22. Contrary to expectations, there was no clear differential effect on developmental timing or bang-sensitivity oftko25t(or wild-type flies) from the knockdown ofCG17192,Fbp1,Fbp2orImpL3. However,ImpL3-targeted RNAi was lethal (tko25tand wild-type), RNAi againstFbp1orFbp2resulted in partial lethality, whereasCG17192-targeted RNAi gave diverse results. The GeneSwitch system influenced developmental processes in bothtko25tand wild-type flies.
Conclusions: Specific, altered nutritional needs of flies with OXPHOS dysfunction were not revealed. The diminished mitochondrial translational capacity and the associated OXPHOS deficiency appear to have a greater effect on expression than the nutrient composition of the diet. Although down-regulation does not have a specific effect on the most substantial phenotypic characteristics oftko25t, the genes of interest seem to perform essential functions inD. melanogaster. It is conceivable that the existence of complementary/redundant metabolic pathways affects the results. However, more research is needed to interpret unequivocally if the expressional changes detected intko25tare adaptive or maladaptive to mitochondrial dysfunction. Although commonly accepted and widely used inD. melanogasterresearch, the GeneSwitch system might not be absolutely reliable.
Methods: Nutritional effects on expression up-regulation were tested on four different culture media, with variable sugar and protein content, and measured using Q-RT-PCR. Drug-inducible GeneSwitch (GAL4-UAS) expression system and transgenicD. melanogasterRNAi lines were used for knockdown. The phenotypic effects of expression down-regulation were determined by performing developmental time and bang-sensitivity experiments. Bothtko25tand wild-type flies were studied.
Results: All the genes tested were up-regulated intko25ton all diets and in both sexes, compared to wild-type flies. In general, there were no major effects of diet, and the transcriptional response was quantitatively similar on all diets. With few exceptions, slightly increased expression levels were observed in wild-type flies grown on low-sugar diets. Functional RNAi lines were not found forCG11659orHsp22. Contrary to expectations, there was no clear differential effect on developmental timing or bang-sensitivity oftko25t(or wild-type flies) from the knockdown ofCG17192,Fbp1,Fbp2orImpL3. However,ImpL3-targeted RNAi was lethal (tko25tand wild-type), RNAi againstFbp1orFbp2resulted in partial lethality, whereasCG17192-targeted RNAi gave diverse results. The GeneSwitch system influenced developmental processes in bothtko25tand wild-type flies.
Conclusions: Specific, altered nutritional needs of flies with OXPHOS dysfunction were not revealed. The diminished mitochondrial translational capacity and the associated OXPHOS deficiency appear to have a greater effect on expression than the nutrient composition of the diet. Although down-regulation does not have a specific effect on the most substantial phenotypic characteristics oftko25t, the genes of interest seem to perform essential functions inD. melanogaster. It is conceivable that the existence of complementary/redundant metabolic pathways affects the results. However, more research is needed to interpret unequivocally if the expressional changes detected intko25tare adaptive or maladaptive to mitochondrial dysfunction. Although commonly accepted and widely used inD. melanogasterresearch, the GeneSwitch system might not be absolutely reliable.