5-Aminolevulinic acid bypasses mitochondrial complex I deficiency and corrects physiological dysfunctions in Drosophila

Abstract

Complex I (CI) deficiency in mitochondrial oxidative phosphorylation (OXPHOS) is the most common cause of mitochondrial diseases, and limited evidence-based treatment options exist. Although CI provides the most electrons to OXPHOS, complex II (CII) is another entry point of electrons. Enhancement of this pathway may compensate for a loss of CI; however, the effects of boosting CII activity on CI deficiency are unclear at the animal level. 5-Aminolevulinic acid (5-ALA) is a crucial precursor of heme, which is essential for CII, complex III, complex IV (CIV) and cytochrome c activities. Here, we show that feeding a combination of 5-ALA hydrochloride and sodium ferrous citrate (5-ALA-HCl + SFC) increases ATP production and suppresses defective phenotypes in Drosophila with CI deficiency. Knockdown of sicily, a Drosophila homolog of the critical CI assembly protein NDUFAF6, caused CI deficiency, accumulation of lactate and pyruvate and detrimental phenotypes such as abnormal neuromuscular junction development, locomotor dysfunctions and premature death. 5-ALA-HCl + SFC feeding increased ATP levels without recovery of CI activity. The activities of CII and CIV were upregulated, and accumulation of lactate and pyruvate was suppressed. 5-ALA-HCl + SFC feeding improved neuromuscular junction development and locomotor functions in sicily-knockdown flies. These results suggest that 5-ALA-HCl + SFC shifts metabolic programs to cope with CI deficiency. Bullet outline 5-Aminolevulinic acid (5-ALA-HCl + SFC) increases ATP production in flies with complex I deficiency.5-ALA-HCl + SFC increases the activities of complexes II and IV.5-ALA-HCl + SFC corrects metabolic abnormalities and suppresses the detrimental phenotypes caused by complex I deficiency.

Figure 1

sicily knockdown causes a developmental delay and neurological defects. (A) Expression of sicily RNAi lowered sicily mRNA expression. Homogenates of adult flies with sicily RNAi expression driven by the ubiquitous driver actin-GAL4 were subjected to RT-qPCR. Flies expressing mCherry RNAi were used as a control (mean ± SE, n = 3; ^****P < 0.005; unpaired t-test). (B) NMJs of third instar larvae were costained with phalloidin (red) and an anti-HRP antibody (green), which recognizes a neural membrane epitope. Genotypes or conditions as indicated. All scale bars, 100 μm. NMJ growth was assessed by counting the number of branches and boutons at abdominal segment A4, muscle 5 (mean ± SE, n = 4; P = 0.05; unpaired t-test). (C) The distance that larvae traveled to form pupae was assessed by counting the number of pupae formed in the top, middle and bottom areas of the vial wall (mean ± SE, n = 57 or 100; ^***P = 0.005; Chi-square test). (D) Egg-to-adult eclosion frequency for progeny flies generated by the cross actin-GAL4/TM3Sb × UAS-sicily RNAi/TM3Ser. Eclosion frequency of actin-GAL4;TM3Ser (control) or actin-GAL4:sicily RNAi (sicily RNAi) expressed as relative ratios (n = 873–1157; ^****P < 0.001; Chi-square test). (E) sicily knockdown caused locomotor deficits. Flies were subjected to a climbing assay; they were tapped to the bottom of the vial, and the distance that they climbed in 10 s was measured. Flies were at 12 days after eclosion (mean ± SE, n = 6–29; ^****P < 0.001; unpaired t-test). (F) Aged females with sicily knockdown developed shock-induced paralysis. The amount of time taken to recover from mechanical stress (bang sensitivity) is shown for flies of the indicated genotype at 24 days after eclosion (mean ± SE, n = 9 or 11; ^**P < 0.01; unpaired t-test). (G) Kaplan–Meier survival curves of sicily-knockdown males and females. Numbers in parentheses indicate sample size (number of flies). ^****P < 0.001, log-rank test.

Figure 2

Effect of sicily knockdown on mitochondrial respiratory complexes. (A) The numbers of mitochondrial DNA copies in thoraxes dissected from 3-day-old flies expressing sicily RNAi and mCherry RNAi (control RNAi) were assessed by qRT-PCR) and are shown as ratios relative to control RNAi (mean ± SE, n  =  3). No significant difference was detected (P > 0.05; unpaired t-test). (B) Heatmap showing fold changes in mRNA levels of the indicated genes (n = 3; ^*P < 0.05; unpaired t-test). (C) Blue-native PAGE and in-gel activity assay of mitochondria extracted from thoraxes of 3-day-old flies of the indicated genotype and sex. Arrows indicate bands specific to sicily knockdown. Asterisks indicate a possible CI fragment. Numbers in parentheses indicate the band intensities in flies expressing sicily RNAi relative to those in flies expressing control RNAi. (D) Heatmap showing fold changes in mRNA levels of the indicated antioxidant genes (n = 3; ^*P < 0.05; unpaired t-test).

Figure 3

sicily knockdown causes lactate and pyruvate accumulation. (A-B) Relative abundance of lactate (A) and pyruvate (B) in flies of the indicated genotype and sex (mean ± SE, n = 3–4; ^***P < 0.005, ^****P < 0.0001; unpaired t-test). (C) Heatmap showing fold changes in mRNA levels of metabolic genes (n = 3; ^*P < 0.05; unpaired t-test).

Figure 4

5-ALA-HCl + SFC feeding increases ATP levels without CI. (A) Quantitation of the ATP levels in thoraxes of sicily-knockdown flies fed food containing the indicated concentrations of 5-ALA-HCl + SFC. Flies were 3 days old. ATP levels are normalized to protein levels and shown as ratios relative to the control (mean ± SD, n  =  3; ^***P < 0.005, ^****P = 0.001; unpaired t-test). (B) The number of mitochondrial copies was determined by qRT-PCR. The numbers of mitochondrial copies are shown as ratios relative to the control (mean ± SE, n  = 3). No significant difference was detected (P > 0.05, unpaired t-test). (C) Heatmap showing fold changes in mRNA levels of the indicated genes (n = 3; ^*P < 0.05, unpaired t-test). (D) Blue-native PAGE and an in-gel activity assay of mitochondria extracted from thoraxes of flies fed food containing the indicated concentrations of 5-ALA-HCl + SFC. Flies were at 3 days after eclosion. Numbers in parentheses indicate the band intensities in flies expressing sicily RNAi relative to those in flies expressing control RNAi.

Figure 5

5-ALA-HCl + SFC suppresses lactate and pyruvate accumulation caused by sicily knockdown. (A-B) Relative abundance of lactate (A) and pyruvate (B) in sicily-knockdown flies fed food containing the indicated concentrations of 5-ALA-HCl + SFC (mean ± SE, n = 3–4; ^**P < 0.01, ^****P < 0.0001; unpaired t-test). (C) Heatmap showing fold changes in mRNA levels of metabolic genes (n = 3; ^*P < 0.05; unpaired t-test).

Figure 6

5-ALA-HCl + SFC feeding rescues developmental delays, premature death and neurological dysfunction caused by sicily knockdown. (A) Third instar larvae were costained with phalloidin (red) and an anti-HRP antibody (green), which recognizes a neural membrane epitope. Concentrations of 5-ALA-HCl + SFC in food are as indicated. NMJ growth was assessed by counting the numbers of branches and boutons at abdominal segment A4, muscle 6/7 (mean ± SE, n = 4; ^**P = 0.01, ^***P = 0.005; unpaired t-test). (B) Egg-to-adult eclosion frequency of flies with the indicated concentrations of 5-ALA-HCl + SFC in food (n = 74–115; ^**P < 0.01; Chi-square test and Holm–Sidak post hoc test). (C) Locomotor functions of flies fed food containing the indicated concentrations of 5-ALA-HCl + SFC were assessed by a climbing assay. Flies were tapped to the bottom of the vial, and the number of flies that reached the top, middle and bottom third of the vial in 20 s was counted. Flies were at 9 days after eclosion (mean ± SE, n = 4–29; ^*P < 0.05, ^****P < 0.0001; one-way ANOVA followed by Dunnett’s test). (D) Amount of time taken to recover from mechanical stress (bang sensitivity) by females fed food containing the indicated concentrations of 5-ALA-HCl + SFC. Flies were at 21 days after eclosion (mean ± SE, n = 11–26; ^*P < 0.05, ^**P < 0.01; one-way ANOVA followed by Dunnett’s test). (E) Survival curves of sicily-knockdown males and females raised on food containing the indicated concentrations of 5-ALA-HCl + SFC. Numbers in parentheses indicate sample size (number of flies); ^*P < 0.05; log-rank test and Holm–Sidak post hoc test. (F–H) 5-ALA-HCl + SFC feeding after eclosion suppressed detrimental phenotypes caused by sicily knockdown. Flies were raised on food lacking 5-ALA-HCl + SFC until eclosion and then transferred to food containing the indicated concentrations of 5-ALA-HCl + SFC. (F) Climbing assay with flies fed food containing the indicated concentrations of 5-ALA-HCl + SFC. Flies were at 12 days after eclosion (mean ± SE, n = 10–22; ^**P < 0.01, ^****P < 0.001; one-way ANOVA followed by Dunnett’s test). (G) Amount of time taken to recover from mechanical stress by females fed food containing the indicated concentrations of 5-ALA-HCl + SFC. Flies were at 30 days after eclosion (mean ± SE, n = 13–30; ^**P < 0.01; one-way ANOVA followed by Dunnett’s test). (H) Survival curves of sicily-knockdown males and females maintained on food containing the indicated concentrations of 5-ALA-HCl + SFC after eclosion. Numbers in parentheses indicate sample size (number of flies); ^*P < 0.05, ^***P < 0.005; log-rank test and Holm–Sidak post hoc test.

Figure 7

Possible mechanisms by which 5-ALA-HCl + SFC increases ATP production without CI. The components upregulated and downregulated by 5-ALA-HCl + SFC are colored orange and blue, respectively. Sicily knockdown causes loss of CI, whereas upregulation of CII compensates for the lack of electron transfer in CI. Upregulation of CIII and CIV increases the number of protons pumped into the IMS. 5-ALA-HCl + SFC may also affect other ATP-synthesizing reactions, including glycolysis, the TCA cycle and β-oxidation, which in combination may counteract the accumulation of lactate and pyruvate and increase ATP synthesis.