Phenotypic rescue of a Drosophila model of mitochondrial ANT1 disease

Abstract

A point mutation in the Drosophila gene that codes for the major adult isoform of adenine nuclear translocase (ANT) represents a model for human diseases that are associated with ANT insufficiency [stress-sensitive B(1) (sesB(1))]. We characterized the organismal, bioenergetic and molecular phenotype of sesB(1) flies then tested strategies to compensate the mutant phenotype. In addition to developmental delay and mechanical-stress-induced seizures, sesB(1) flies have an impaired response to sound, defective male courtship, female sterility and curtailed lifespan. These phenotypes, excluding the latter two, are shared with the mitoribosomal protein S12 mutant, tko(25t). Mitochondria from sesB(1) adults showed a decreased respiratory control ratio and downregulation of cytochrome oxidase. sesB(1) adults exhibited ATP depletion, lactate accumulation and changes in gene expression that were consistent with a metabolic shift towards glycolysis, characterized by activation of lactate dehydrogenase and anaplerotic pathways. Females also showed downregulation of many genes that are required for oogenesis, and their eggs, although fertilized, failed to develop to the larval stages. The sesB(1) phenotypes of developmental delay and mechanical-stress-induced seizures were alleviated by an altered mitochondrial DNA background. Female sterility was substantially rescued by somatic expression of alternative oxidase (AOX) from the sea squirt Ciona intestinalis, whereas AOX did not alleviate developmental delay. Our findings illustrate the potential of different therapeutic strategies for ANT-linked diseases, based on alleviating metabolic stress.

Keywords: Adenine nucleotide translocase; Alternative oxidase; Mitochondrial biogenesis; Mitochondrial disease.

Fig. 1.

Genotype and phenotype of sesB¹ flies. (A) Aligned partial sequences of sesB mRNA, isoform A (sesB+), commencing at nucleotide 962 of NCBI database entry NM_078554, and human ANT1 mRNA (NCBI database entry NM_001151 from nucleotide 863). The corresponding amino acid sequences and the base substitutions (red) and corresponding amino acid changes in the sesB¹ allele, verified in the strain analyzed here, are also shown. The ANT1 gene mutation shown here is that reported in an Italian family with adPEO (Kaukonen et al., 2000). Each mutation results in the substitution of a hydrophobic residue in transmembrane segment VI of the protein (Pebray-Peyroula et al., 2003) by another hydrophobic residue. (B) The day of eclosion of flies of the genotypes indicated; means±s.e.m. are shown from five replicate vials. FM7 is the X-chromosomal balancer chromosome used to maintain heterozygosity for sesB¹. (C) The square-root of the time taken to recover from mechanical shock (‘bang sensitivity’) for batches of ≥25 flies of each sex and genotype that were individually analyzed; means±s.e.m. are shown. The day of eclosion and bang sensitivity of sesB¹ flies were significantly different from those of wild-type (wt) or heterozygous flies of the same sex (P<0.01). (D) Response of adult males of the indicated strains to sound. The mean number of responders±s.d. amongst groups of six males from three replicate groups of each genotype are shown. Bang-sensitive strains are: tko^25t, point mutant of technical knockout, which encodes mitoribosomal protein S12 (Shah et al., 1997); bss, bang senseless, a point mutant of paralytic, which encodes a voltage-gated Na⁺ channel (Parker et al., 2011); sesB¹; and sda, slamdance, which encodes aminopeptidase N (Zhang et al., 2002). tko^25t, sesB¹ and sda are non-responsive, whereas bss has normal hearing. (E) Lifespan curves for sesB¹ and wild-type control flies of the sexes indicated (groups of 100 flies of each sex and genotype) when maintained at 25°C. Three replicate experiments gave similar results – i.e. mean lifespans of 11–13 days for sesB¹ males and 13–18 days for sesB¹ females. (F) GFP that was targeted to the mitochondria was expressed in indirect flight muscles of wild-type and sesB¹ flies (in both genotypes in the presence of a transgene encoding UAS-mito-HA-GFP and the Mef2-GAL4 driver). The mitochondrial network appears normal in sesB¹. (G) Dark-field micrograph of the dissected ovaries from 2-day-old wild-type and sesB¹ females. (H) Phase-contrast micrograph of developing embryos from eggs laid by sesB¹ females. The vast majority of embryos failed to hatch, and only a tiny number (~0.1%) completed development.

Fig. 2.

Mitochondrial metabolism in sesB¹ flies. (A) Apparent respiratory control ratio (RCR; the relative rate of oxygen consumption before and after the addition of ADP) of isolated mitochondria from flies of the strains and sexes indicated, using different substrate mixes, based on the data shown in Table 1. Note that the value obtained with the pyruvate and proline substrate mix is not a true RCR, because of the secondary activation of isocitrate dehydrogenase by ADP (Miwa et al., 2003). However, a similar trend is observed with both substrate mixes. (B) Steady-state ATP levels in the extracts from flies of the strains and sexes indicated, normalized to the value in wild-type (wt) flies in the given sex. The means of results from five or six biological replicates for each group are shown. (C) Steady-state lactate levels in extracts from 10-day-old flies of strains and sexes indicated. n=6 for males, n=3 for females. (A–C) Means±s.d., *P<0.05, **P<0.01 by using Student’s t-test. (D) Representative blue native electrophoresis gels, which were stained histochemically for complex I and IV, of extracts from flies of the strains and sexes indicated. Arrows indicate the major complexes that were stained; their apparent molecular weights were extrapolated from the migration of markers.

Fig. 3.

Summary of transcriptomic analysis of sesB¹ flies. (A–D) Diagrammatic representation of degree of overlap between probesets that had altered expression from wild type; fly strains and sexes are indicated. For details, see supplementary material Tables S1–S7. (E) GO classification by molecular function of the genes that showed altered expression in sesB¹ flies of both sexes (panel A; supplementary material Table S1).

Fig. 4.

Testing rescue of sesB¹ by altered mtDNA background or spargel overexpression. (A,B) The day of eclosion of flies of the genotypes and sexes indicated is shown; means±s.d. from three replicate experiments. FM7 is the X-chromosomal balancer chromosome used to maintain heterozygosity for sesB¹, which gives a wild-type phenotype. BER-1;sesB¹ denotes cybrid flies (BER-1 mtDNA, sesB¹ mutation and nuclear background). srl^GR (an additional genomic copy of spargel) was homozygous, as indicated. (C) The square-root of the recovery time from mechanical shock (bang sensitivity) of flies of the genotypes and sexes indicated; means±s.e.m. are shown. srl^GR flies in this experiment were hemizygous. The number of individually analyzed flies of each genotype was between 19 and 24 for the different classes. In B and C, ^#P<0.05, *P<0.01 between sesB¹ and other data classes. Note that t-test or ANOVA cannot be meaningfully implemented for A owing to the skewed distribution of the day of eclosion in the BER-1 mtDNA background; hence the data are only indicative. The partial rescue of bang sensitivity by overexpression of spargel is statistically significant, but because an entire chromosome is replaced in this experiment, we cannot exclude a contribution of the genetic background. (D) Relative spargel mRNA levels, in flies of the sex and genotype indicated, normalized to the value in the same sex for wild-type flies. Only the lines that overexpressed spargel showed a significant increase in the mRNA, denoted by *, as above. (E) The mean proportion of eggs that developed into adults for the offspring of females, of the genotypes indicated, mated to wild-type males. Means±s.d. from three replicate experiments are shown.

Fig. 5.

Partial rescue of sesB¹ by AOX expression. (A) The day of eclosion of flies of the genotypes and sexes indicated, reared at 22°C. Where present, da-GAL4 and UAS-AOX transgenes were hemizygous. ** significant differences between sesB¹ and other data classes (P<0.01). The means±s.d. from eight to 14 replicate experiments are shown. (B) Bang sensitivity – the square-root of recovery time from mechanical shock of 3-day-old flies of the genotypes and sexes indicated, reared at 22°C; means±s.e.m. are shown. 79–215 flies of each class were analyzed individually. **P<0.01 between data classes for each sex (calculated by Student’s t-test with Bonferroni correction). (C) Mean numbers of offspring per vial as a result of mating females of the indicated genotypes (reared at 22°C) with wild-type (Oregon R) males. +, hemizygous; ++, homozygous; −, absence of a given marker, balancer or transgene. *P<0.05, **P<0.01 between sesB¹;AOX and other data classes for each sex (Student’s t-test). (D) Lifespan curves for male flies of the indicated genotypes. Canton S (CS) and Oregon R (OR) are different strain backgrounds, which show curtailed lifespans similar to that of sesB¹. Where present together, da-GAL4 and UAS-AOX transgenes were hemizygous. When UAS-AOX alone was present, it was homozygous in order to control for the number of copies of the mini-white selectable marker. The survival curve for AOX-expressing males is significantly different from all of the control lines, including sesB¹ in either genetic background (log-rank test, P<0.001). However, given that the increase in mean lifespan was quantitatively minor, and was not seen in females, a contribution from the genetic background cannot be excluded.

Fig. 6.

Lack of rescue of sesB¹ by Ndi1 expression. (A) The day of eclosion of flies of the genotypes and sexes indicated; means±s.e.m. from three biological replicates are shown. Where present, da-GAL4 and UAS-Ndi1 transgenes were hemizygous. **P<0.01 between sesB¹;Ndi1 and other data classes for each sex (Student’s t-test with Bonferroni correction where appropriate). (B) The mean numbers of offspring per vial resulting from the mating of females of the genotypes indicated with wild-type (Oregon R) males. +, hemizygous; ++, homozygous; −, absence of a given marker, balancer or transgene.