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dATF4 regulation of mitochondrial folate-mediated one-carbon metabolism is neuroprotective

View Article: PubMed Central - PubMed

ABSTRACT

Neurons rely on mitochondria as their preferred source of energy. Mutations in PINK1 and PARKIN cause neuronal death in early-onset Parkinson's disease (PD), thought to be due to mitochondrial dysfunction. In Drosophila pink1 and parkin mutants, mitochondrial defects lead to the compensatory upregulation of the mitochondrial one-carbon cycle metabolism genes by an unknown mechanism. Here we uncover that this branch is triggered by the activating transcription factor 4 (ATF4). We show that ATF4 regulates the expression of one-carbon metabolism genes SHMT2 and NMDMC as a protective response to mitochondrial toxicity. Suppressing Shmt2 or Nmdmc caused motor impairment and mitochondrial defects in flies. Epistatic analyses showed that suppressing the upregulation of Shmt2 or Nmdmc deteriorates the phenotype of pink1 or parkin mutants. Conversely, the genetic enhancement of these one-carbon metabolism genes in pink1 or parkin mutants was neuroprotective. We conclude that mitochondrial dysfunction caused by mutations in the Pink1/Parkin pathway engages ATF4-dependent activation of one-carbon metabolism as a protective response. Our findings show a central contribution of ATF4 signalling to PD that may represent a new therapeutic strategy. A video abstract for this article is available at https://youtu.be/cFJJm2YZKKM.

No MeSH data available.


Related in: MedlinePlus

Suppression of Shmt2 or Nmdmc causes mitochondrial defects. (a and b) RNAi-mediated suppression of Shmt2 or Nmdmc results in mitochondrial fragmentation. (a) Confocal analysis of mitoGFP in the indicated larval tissues. (b) The quantification of mitochondrial length from larval nerve is indicated (mean±S.D., P-value, one-way ANOVA with Bonferroni's multiple comparison test). (c) Representative confocal image of a whole mounted control brain showing neurons loaded with TMRM. (d) Loss of Δψm following RNAi-mediated suppression of Shmt2 or Nmdmc. The data are shown as the mean±S.D. (asterisks, one-way ANOVA with Bonferroni's multiple comparison test). (e) Loss of mitochondrial proteins in Shmt2 and Nmdmc RNAi flies. Whole-fly lysates from 3-day-old flies were analysed by western blot analysis using the indicated antibodies. (f) Abnormal wing posture following RNAi-mediated suppression of Nmdmc (asterisks, χ2 two-tailed, 95% confidence intervals). (g) The knockdown of Nmdmc causes mitochondrial cristae fragmentation. Ultrastructural analysis of the indirect flight muscles in Nmdmc RNAi flies (my, myofibrils; m, mitochondria; yellow outlines, mitochondria). Percentages of indirect-flight-muscle mitochondria exhibiting fragmented cristae normalized to area are presented (asterisks, χ2 two-tailed, 95% confidence intervals). Genotype in (a and b): Control: elavGAL4>mitoGFP; Shmt2 RNAi #1: elavGAL4>mitoGFP, Shmt2 RNAi #1; Nmdmc RNAi #1: elavGAL4>mitoGFP, Nmdmc RNAi #1, (c and d): Control: elavGAL4. All RNAi lines were driven by elavGAL4, (e–g): Control: daGAL4. All RNAi lines were driven by daGAL4
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fig4: Suppression of Shmt2 or Nmdmc causes mitochondrial defects. (a and b) RNAi-mediated suppression of Shmt2 or Nmdmc results in mitochondrial fragmentation. (a) Confocal analysis of mitoGFP in the indicated larval tissues. (b) The quantification of mitochondrial length from larval nerve is indicated (mean±S.D., P-value, one-way ANOVA with Bonferroni's multiple comparison test). (c) Representative confocal image of a whole mounted control brain showing neurons loaded with TMRM. (d) Loss of Δψm following RNAi-mediated suppression of Shmt2 or Nmdmc. The data are shown as the mean±S.D. (asterisks, one-way ANOVA with Bonferroni's multiple comparison test). (e) Loss of mitochondrial proteins in Shmt2 and Nmdmc RNAi flies. Whole-fly lysates from 3-day-old flies were analysed by western blot analysis using the indicated antibodies. (f) Abnormal wing posture following RNAi-mediated suppression of Nmdmc (asterisks, χ2 two-tailed, 95% confidence intervals). (g) The knockdown of Nmdmc causes mitochondrial cristae fragmentation. Ultrastructural analysis of the indirect flight muscles in Nmdmc RNAi flies (my, myofibrils; m, mitochondria; yellow outlines, mitochondria). Percentages of indirect-flight-muscle mitochondria exhibiting fragmented cristae normalized to area are presented (asterisks, χ2 two-tailed, 95% confidence intervals). Genotype in (a and b): Control: elavGAL4>mitoGFP; Shmt2 RNAi #1: elavGAL4>mitoGFP, Shmt2 RNAi #1; Nmdmc RNAi #1: elavGAL4>mitoGFP, Nmdmc RNAi #1, (c and d): Control: elavGAL4. All RNAi lines were driven by elavGAL4, (e–g): Control: daGAL4. All RNAi lines were driven by daGAL4

Mentions: To investigate the in vivo role of Shmt2 and Nmdmc, we determined the consequences of their suppression using RNAi (Figure 3a). The knockdown of Shmt2 or Nmdmc caused developmental defects characterized by a significant failure of eclosion (Figure 3b). Analysis of the eclosed adults revealed that the knockdown of Shmt2 or Nmdmc resulted in an impaired climbing ability, suggesting a locomotor deficit (Figure 3c), and decreased lifespan (Figure 3d). The knockdown of either Shmt2 or Nmdmc led to significant metabolic changes in several canonical pathways, most significantly those related to nucleotide degradation and salvage (Figure 3e and Supplementary Table 2). To further determine whether the consequences of Shmt2 or Nmdmc knockdown were linked to mitochondrial defects, we performed a morphological and functional analysis of mitochondria. This revealed a fragmented mitochondrial network (Figures 4a and b) that was associated with a loss of mitochondrial membrane potential (Δψm) in adult brain (Figures 4c and d) as well as a generalized loss of mitochondrial proteins (Figure 4e). In addition, Nmdmc knockdown adult flies exhibited an abnormal downturned wing posture (Figure 4f) and ultrastructural analysis of their indirect flight muscles revealed mitochondria with fragmented cristae (Figure 4g).


dATF4 regulation of mitochondrial folate-mediated one-carbon metabolism is neuroprotective
Suppression of Shmt2 or Nmdmc causes mitochondrial defects. (a and b) RNAi-mediated suppression of Shmt2 or Nmdmc results in mitochondrial fragmentation. (a) Confocal analysis of mitoGFP in the indicated larval tissues. (b) The quantification of mitochondrial length from larval nerve is indicated (mean±S.D., P-value, one-way ANOVA with Bonferroni's multiple comparison test). (c) Representative confocal image of a whole mounted control brain showing neurons loaded with TMRM. (d) Loss of Δψm following RNAi-mediated suppression of Shmt2 or Nmdmc. The data are shown as the mean±S.D. (asterisks, one-way ANOVA with Bonferroni's multiple comparison test). (e) Loss of mitochondrial proteins in Shmt2 and Nmdmc RNAi flies. Whole-fly lysates from 3-day-old flies were analysed by western blot analysis using the indicated antibodies. (f) Abnormal wing posture following RNAi-mediated suppression of Nmdmc (asterisks, χ2 two-tailed, 95% confidence intervals). (g) The knockdown of Nmdmc causes mitochondrial cristae fragmentation. Ultrastructural analysis of the indirect flight muscles in Nmdmc RNAi flies (my, myofibrils; m, mitochondria; yellow outlines, mitochondria). Percentages of indirect-flight-muscle mitochondria exhibiting fragmented cristae normalized to area are presented (asterisks, χ2 two-tailed, 95% confidence intervals). Genotype in (a and b): Control: elavGAL4>mitoGFP; Shmt2 RNAi #1: elavGAL4>mitoGFP, Shmt2 RNAi #1; Nmdmc RNAi #1: elavGAL4>mitoGFP, Nmdmc RNAi #1, (c and d): Control: elavGAL4. All RNAi lines were driven by elavGAL4, (e–g): Control: daGAL4. All RNAi lines were driven by daGAL4
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fig4: Suppression of Shmt2 or Nmdmc causes mitochondrial defects. (a and b) RNAi-mediated suppression of Shmt2 or Nmdmc results in mitochondrial fragmentation. (a) Confocal analysis of mitoGFP in the indicated larval tissues. (b) The quantification of mitochondrial length from larval nerve is indicated (mean±S.D., P-value, one-way ANOVA with Bonferroni's multiple comparison test). (c) Representative confocal image of a whole mounted control brain showing neurons loaded with TMRM. (d) Loss of Δψm following RNAi-mediated suppression of Shmt2 or Nmdmc. The data are shown as the mean±S.D. (asterisks, one-way ANOVA with Bonferroni's multiple comparison test). (e) Loss of mitochondrial proteins in Shmt2 and Nmdmc RNAi flies. Whole-fly lysates from 3-day-old flies were analysed by western blot analysis using the indicated antibodies. (f) Abnormal wing posture following RNAi-mediated suppression of Nmdmc (asterisks, χ2 two-tailed, 95% confidence intervals). (g) The knockdown of Nmdmc causes mitochondrial cristae fragmentation. Ultrastructural analysis of the indirect flight muscles in Nmdmc RNAi flies (my, myofibrils; m, mitochondria; yellow outlines, mitochondria). Percentages of indirect-flight-muscle mitochondria exhibiting fragmented cristae normalized to area are presented (asterisks, χ2 two-tailed, 95% confidence intervals). Genotype in (a and b): Control: elavGAL4>mitoGFP; Shmt2 RNAi #1: elavGAL4>mitoGFP, Shmt2 RNAi #1; Nmdmc RNAi #1: elavGAL4>mitoGFP, Nmdmc RNAi #1, (c and d): Control: elavGAL4. All RNAi lines were driven by elavGAL4, (e–g): Control: daGAL4. All RNAi lines were driven by daGAL4
Mentions: To investigate the in vivo role of Shmt2 and Nmdmc, we determined the consequences of their suppression using RNAi (Figure 3a). The knockdown of Shmt2 or Nmdmc caused developmental defects characterized by a significant failure of eclosion (Figure 3b). Analysis of the eclosed adults revealed that the knockdown of Shmt2 or Nmdmc resulted in an impaired climbing ability, suggesting a locomotor deficit (Figure 3c), and decreased lifespan (Figure 3d). The knockdown of either Shmt2 or Nmdmc led to significant metabolic changes in several canonical pathways, most significantly those related to nucleotide degradation and salvage (Figure 3e and Supplementary Table 2). To further determine whether the consequences of Shmt2 or Nmdmc knockdown were linked to mitochondrial defects, we performed a morphological and functional analysis of mitochondria. This revealed a fragmented mitochondrial network (Figures 4a and b) that was associated with a loss of mitochondrial membrane potential (Δψm) in adult brain (Figures 4c and d) as well as a generalized loss of mitochondrial proteins (Figure 4e). In addition, Nmdmc knockdown adult flies exhibited an abnormal downturned wing posture (Figure 4f) and ultrastructural analysis of their indirect flight muscles revealed mitochondria with fragmented cristae (Figure 4g).

View Article: PubMed Central - PubMed

ABSTRACT

Neurons rely on mitochondria as their preferred source of energy. Mutations in PINK1 and PARKIN cause neuronal death in early-onset Parkinson's disease (PD), thought to be due to mitochondrial dysfunction. In Drosophila pink1 and parkin mutants, mitochondrial defects lead to the compensatory upregulation of the mitochondrial one-carbon cycle metabolism genes by an unknown mechanism. Here we uncover that this branch is triggered by the activating transcription factor 4 (ATF4). We show that ATF4 regulates the expression of one-carbon metabolism genes SHMT2 and NMDMC as a protective response to mitochondrial toxicity. Suppressing Shmt2 or Nmdmc caused motor impairment and mitochondrial defects in flies. Epistatic analyses showed that suppressing the upregulation of Shmt2 or Nmdmc deteriorates the phenotype of pink1 or parkin mutants. Conversely, the genetic enhancement of these one-carbon metabolism genes in pink1 or parkin mutants was neuroprotective. We conclude that mitochondrial dysfunction caused by mutations in the Pink1/Parkin pathway engages ATF4-dependent activation of one-carbon metabolism as a protective response. Our findings show a central contribution of ATF4 signalling to PD that may represent a new therapeutic strategy. A video abstract for this article is available at https://youtu.be/cFJJm2YZKKM.

No MeSH data available.


Related in: MedlinePlus