Gene expression in a Drosophila model of mitochondrial disease

Abstract

Background: A point mutation in the Drosophila gene technical knockout (tko), encoding mitoribosomal protein S12, was previously shown to cause a phenotype of respiratory chain deficiency, developmental delay, and neurological abnormalities similar to those presented in many human mitochondrial disorders, as well as defective courtship behavior.

Methodology/principal findings: Here, we describe a transcriptome-wide analysis of gene expression in tko(25t) mutant flies that revealed systematic and compensatory changes in the expression of genes connected with metabolism, including up-regulation of lactate dehydrogenase and of many genes involved in the catabolism of fats and proteins, and various anaplerotic pathways. Gut-specific enzymes involved in the primary mobilization of dietary fats and proteins, as well as a number of transport functions, were also strongly up-regulated, consistent with the idea that oxidative phosphorylation OXPHOS dysfunction is perceived physiologically as a starvation for particular biomolecules. In addition, many stress-response genes were induced. Other changes may reflect a signature of developmental delay, notably a down-regulation of genes connected with reproduction, including gametogenesis, as well as courtship behavior in males; logically this represents a programmed response to a mitochondrially generated starvation signal. The underlying signalling pathway, if conserved, could influence many physiological processes in response to nutritional stress, although any such pathway involved remains unidentified.

Conclusions/significance: These studies indicate that general and organ-specific metabolism is transformed in response to mitochondrial dysfunction, including digestive and absorptive functions, and give important clues as to how novel therapeutic strategies for mitochondrial disorders might be developed.

Figure 1. Crossing scheme to generate maximally outbred tko^25t mutant flies for analysis.

Balanced stocks were used first to create homozygous females and hemizygous males of the two parental backgrounds, in order to include in the analysis any maternal effects of the mutation. Note that tko is an X-chromosomal gene. The initial outbreeding to create the balanced stocks restores a wild-type genetic background, but does not completely eliminate any potentially compensatory recessive alleles already in the wild-type backgrounds. To minimize the effects of any such alleles, the crossing scheme illustrated is both maximally wild-type and heterozygous, under which conditions we saw the most substantial accentuation of the mutant phenotype, compared with inbred tko^25t lines .

Figure 2. Wolbachia infection does not explain the abnormal metabolism or courtship behaviour of tko^25t flies.

PCR reactions analysed on agarose gels, using Wolbachia-specific 16S rDNA and Drosophila mitochondrial 12S rDNA primers. The 897 bp Wolbachia-specific product (arrowed) is detected only in the Wolbachia-infected strain obtained from the Bloomington Stock Center (wol), and not in wild-type (+) or tko^25t flies in either the Oregon R (OR) or Canton S (CS) backgrounds, nor in inbred laboratory stocks of the sesB¹ or to¹ mutants. The 180 bp mitochondrial DNA product is evident in all strains tested. M, 1 kb marker ladder.

Figure 3. Reproductive defects of outbred tko^25t females.

(a) The number of eggs laid per mated female was counted daily for individual mated females from the crosses indicated. Asterisks indicate significant differences (p<0.01, t-test). (b) Single mating pairs of the genotypes shown were observed for time to copulation.

Figure 4. Quantitative RT-PCR verification of transcriptomic data on takeout.

RNA measurements were made and normalized as described in Materials and Methods. Means±SD of three sample runs of each of three biological replicates are shown. Significance at the p value shown was computed using a t-test. See also Table 3.

Figure 5. Summary of major alterations to gene expression and their proposed effects in tko^25t flies.

(a, b) Proposed metabolic effects, based on differences in gene expression affecting nutrition and metabolism between (a) wild-type and (b) tko^25t flies. In wild-type flies glucose is metabolized via PEP to pyruvate, which is then fed to the TCA cycle mainly via the pyruvate dehydrogenase complex generating acetyl-CoA, with a small amount converted to oxaloacetate to replenish the TCA cycle intermediates as needed, maintaining a supply of carbon skeletons for biosynthesis. Surplus NADH is reoxidized via the ETC (complexes I, III and IV), generating potentially most of the cell's ATP needs at complex V. In tko^25t flies, the maximal activity of the ETC complexes is only 10–20% that of wild-type flies . For simplicity, its greatly decreased contribution to NADH oxidation and ATP generation is omitted altogether in panel (b). Instead, the bulk of ATP must be supplied by glycolysis, with NADH reoxidation dependent on lactate dehydrogenase and similar shunts. Because pyruvate is, under such conditions, mainly shunted to lactate, the TCA cycle must be supplied from other sources, via the mobilization of dietary lipids, generating acetyl-CoA, PEP carboxykinase (I) diverting a small amount of PEP to oxaloacetate, and the mobilization of dietary protein and amino acid catabolism supplying these and other TCA cycle intermediates, as well as biosynthetic reactions directly. The modifications to metabolism in tko^25t flies are accompanied (c) by altered expression of genes connected with nutrient breakdown, absorption and transport, plus xenobiotic handling, affecting mainly the gut, Malpighian tubule and fat body. In addition, there is downregulation or delayed expression of genes connected with gametogenesis and skeletogenesis, and, notably in males, altered expression of genes controlling circadian and courtship behaviour, interpretable as a biological response to poor nutritional conditions.