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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

Suppressing the upregulation of Shmt2 or Nmdmc deteriorates the phenotype of pink1 or parkin mutants. (a) RNAi-mediated suppression of dAtf4 enhances lethality in parkin and pink1 mutants (asterisks, χ2 two-tailed, 95% confidence intervals). (b) RNAi-mediated suppression of dAtf4 in pink1 or parkin mutants. Expression levels were measured by real-time qPCR (mean±S.D.). The significance is indicated (asterisks, two-tailed unpaired t-test). (c) Representative images of normal and defective thorax in pink1 mutant, arrow points to a thoracic defect. (d) Enhancement of the thoracic defects in pink1 or parkin mutants by RNAi-mediated suppression of dAtf4 (asterisks, χ2 two-tailed, 95% confidence intervals). (e) Enhancement of the motor impairment of pink1 mutants by RNAi-mediated suppression of dAtf4. Flies were tested using a standard climbing assay (mean±S.D.; asterisks, one-way ANOVA with Bonferroni's multiple comparison test). (f and g) RNAi-mediated suppression of dAtf4 in pink1 or parkin mutants affects the transcript levels of Shmt2 (f) and Nmdmc (g). Expression levels were measured by real-time qPCR (mean±S.D.). The significance is indicated (P-value, two-tailed unpaired t-test compared with pink1B9 or park25). (h) RNAi-mediated suppression of Shmt2 or Nmdmc enhances lethality in parkin mutants (asterisks, χ2 two-tailed, 95% confidence intervals). (i) RNAi-mediated suppression of Shmt2 or Nmdmc in pink1 mutants. Expression levels were measured by real-time qPCR (mean±S.D.). The significant values relative to the control are indicated (asterisks, two-tailed unpaired t-test compared with Control). (j) RNAi-mediated suppression of Shmt2 or Nmdmc enhances lethality in pink1 mutants (asterisks, χ2 two-tailed, 95% confidence intervals). (k) Enhancement of the thoracic defects of pink1 mutants by RNAi-mediated suppression of Shmt2 or Nmdmc (asterisks, χ2 two-tailed, 95% confidence intervals). (l) Enhancement of the motor impairment of pink1 mutants by RNAi-mediated suppression of Nmdmc. Flies were tested using a standard climbing assay (mean±S.D.; asterisks, one-way ANOVA with Bonferroni's multiple comparison test). Genotypes in (a and b and d–l): Control: daGAL4; park25: park25, daGAL4/park25; pink1B9: pink1B9, daGAL4. All RNAi lines were driven by daGAL4. (c): Control: w1118
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fig5: Suppressing the upregulation of Shmt2 or Nmdmc deteriorates the phenotype of pink1 or parkin mutants. (a) RNAi-mediated suppression of dAtf4 enhances lethality in parkin and pink1 mutants (asterisks, χ2 two-tailed, 95% confidence intervals). (b) RNAi-mediated suppression of dAtf4 in pink1 or parkin mutants. Expression levels were measured by real-time qPCR (mean±S.D.). The significance is indicated (asterisks, two-tailed unpaired t-test). (c) Representative images of normal and defective thorax in pink1 mutant, arrow points to a thoracic defect. (d) Enhancement of the thoracic defects in pink1 or parkin mutants by RNAi-mediated suppression of dAtf4 (asterisks, χ2 two-tailed, 95% confidence intervals). (e) Enhancement of the motor impairment of pink1 mutants by RNAi-mediated suppression of dAtf4. Flies were tested using a standard climbing assay (mean±S.D.; asterisks, one-way ANOVA with Bonferroni's multiple comparison test). (f and g) RNAi-mediated suppression of dAtf4 in pink1 or parkin mutants affects the transcript levels of Shmt2 (f) and Nmdmc (g). Expression levels were measured by real-time qPCR (mean±S.D.). The significance is indicated (P-value, two-tailed unpaired t-test compared with pink1B9 or park25). (h) RNAi-mediated suppression of Shmt2 or Nmdmc enhances lethality in parkin mutants (asterisks, χ2 two-tailed, 95% confidence intervals). (i) RNAi-mediated suppression of Shmt2 or Nmdmc in pink1 mutants. Expression levels were measured by real-time qPCR (mean±S.D.). The significant values relative to the control are indicated (asterisks, two-tailed unpaired t-test compared with Control). (j) RNAi-mediated suppression of Shmt2 or Nmdmc enhances lethality in pink1 mutants (asterisks, χ2 two-tailed, 95% confidence intervals). (k) Enhancement of the thoracic defects of pink1 mutants by RNAi-mediated suppression of Shmt2 or Nmdmc (asterisks, χ2 two-tailed, 95% confidence intervals). (l) Enhancement of the motor impairment of pink1 mutants by RNAi-mediated suppression of Nmdmc. Flies were tested using a standard climbing assay (mean±S.D.; asterisks, one-way ANOVA with Bonferroni's multiple comparison test). Genotypes in (a and b and d–l): Control: daGAL4; park25: park25, daGAL4/park25; pink1B9: pink1B9, daGAL4. All RNAi lines were driven by daGAL4. (c): Control: w1118

Mentions: To confirm that transcripts for the one-carbon metabolism genes Shmt2 and Nmdmc are induced in pink1 and parkin mutants as a compensatory mechanism for mitochondrial dysfunction, we tested the consequences of their suppression in these mutants. As ATF4 is required for the upregulation of SHMT2 and NMDMC in mammalian cells, we first determined the in vivo consequences of suppressing its expression in pink1 or parkin mutant flies. The knockdown of dAtf4 led to 11% and 84% lethality, respectively, in pink1 and parkin mutants (Figure 5a). Analysis of the surviving pink1 and parkin mutant adults with dAtf4 knockdown confirmed the downregulation of the dAtf4 transcript (Figure 5b) and revealed an increased penetrance of the crushed-thorax phenotype (Figures 5c and d). In addition, dAtf4 knockdown caused a decline in the climbing performance of pink1 mutants (Figure 5e). We next assessed the effect of dAtf4 knockdown on the levels of Shmt2 and Nmdmc in pink1 and parkin mutants. This caused a moderate decrease in Shmt2 and a more pronounced decrease in Nmdmc transcript levels (Figures 5f and g). Taken together, these results show that dAtf4 is involved in the upregulation of Shmt2 and Nmdmc in pink1 and parkin mutants. Next, we tested the effects of downregulating Shmt2 or Nmdmc in pink1 or parkin mutants. The knockdown of Shmt2 or Nmdmc led to 100% and 99% lethality, respectively, in parkin mutants (Figure 5h). In pink1 mutants, the knockdown of Shmt2 or Nmdmc reduced their respective transcript levels (Figure 5i), and caused 84 and 19% lethality, respectively (Figure 5j). Analysis of the surviving adults revealed an increased penetrance of the crushed-thorax phenotype in both Shmt2 and Nmdmc knockdown (Figure 5k). Furthermore, Nmdmc knockdown exhibited a dramatic decline in the climbing performance of pink1 mutants (Figure 5l). These results suggest that the one-carbon metabolism genes, Shmt2 and Nmdmc, were upregulated as a protective mechanism in pink1 and parkin mutants.


dATF4 regulation of mitochondrial folate-mediated one-carbon metabolism is neuroprotective
Suppressing the upregulation of Shmt2 or Nmdmc deteriorates the phenotype of pink1 or parkin mutants. (a) RNAi-mediated suppression of dAtf4 enhances lethality in parkin and pink1 mutants (asterisks, χ2 two-tailed, 95% confidence intervals). (b) RNAi-mediated suppression of dAtf4 in pink1 or parkin mutants. Expression levels were measured by real-time qPCR (mean±S.D.). The significance is indicated (asterisks, two-tailed unpaired t-test). (c) Representative images of normal and defective thorax in pink1 mutant, arrow points to a thoracic defect. (d) Enhancement of the thoracic defects in pink1 or parkin mutants by RNAi-mediated suppression of dAtf4 (asterisks, χ2 two-tailed, 95% confidence intervals). (e) Enhancement of the motor impairment of pink1 mutants by RNAi-mediated suppression of dAtf4. Flies were tested using a standard climbing assay (mean±S.D.; asterisks, one-way ANOVA with Bonferroni's multiple comparison test). (f and g) RNAi-mediated suppression of dAtf4 in pink1 or parkin mutants affects the transcript levels of Shmt2 (f) and Nmdmc (g). Expression levels were measured by real-time qPCR (mean±S.D.). The significance is indicated (P-value, two-tailed unpaired t-test compared with pink1B9 or park25). (h) RNAi-mediated suppression of Shmt2 or Nmdmc enhances lethality in parkin mutants (asterisks, χ2 two-tailed, 95% confidence intervals). (i) RNAi-mediated suppression of Shmt2 or Nmdmc in pink1 mutants. Expression levels were measured by real-time qPCR (mean±S.D.). The significant values relative to the control are indicated (asterisks, two-tailed unpaired t-test compared with Control). (j) RNAi-mediated suppression of Shmt2 or Nmdmc enhances lethality in pink1 mutants (asterisks, χ2 two-tailed, 95% confidence intervals). (k) Enhancement of the thoracic defects of pink1 mutants by RNAi-mediated suppression of Shmt2 or Nmdmc (asterisks, χ2 two-tailed, 95% confidence intervals). (l) Enhancement of the motor impairment of pink1 mutants by RNAi-mediated suppression of Nmdmc. Flies were tested using a standard climbing assay (mean±S.D.; asterisks, one-way ANOVA with Bonferroni's multiple comparison test). Genotypes in (a and b and d–l): Control: daGAL4; park25: park25, daGAL4/park25; pink1B9: pink1B9, daGAL4. All RNAi lines were driven by daGAL4. (c): Control: w1118
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fig5: Suppressing the upregulation of Shmt2 or Nmdmc deteriorates the phenotype of pink1 or parkin mutants. (a) RNAi-mediated suppression of dAtf4 enhances lethality in parkin and pink1 mutants (asterisks, χ2 two-tailed, 95% confidence intervals). (b) RNAi-mediated suppression of dAtf4 in pink1 or parkin mutants. Expression levels were measured by real-time qPCR (mean±S.D.). The significance is indicated (asterisks, two-tailed unpaired t-test). (c) Representative images of normal and defective thorax in pink1 mutant, arrow points to a thoracic defect. (d) Enhancement of the thoracic defects in pink1 or parkin mutants by RNAi-mediated suppression of dAtf4 (asterisks, χ2 two-tailed, 95% confidence intervals). (e) Enhancement of the motor impairment of pink1 mutants by RNAi-mediated suppression of dAtf4. Flies were tested using a standard climbing assay (mean±S.D.; asterisks, one-way ANOVA with Bonferroni's multiple comparison test). (f and g) RNAi-mediated suppression of dAtf4 in pink1 or parkin mutants affects the transcript levels of Shmt2 (f) and Nmdmc (g). Expression levels were measured by real-time qPCR (mean±S.D.). The significance is indicated (P-value, two-tailed unpaired t-test compared with pink1B9 or park25). (h) RNAi-mediated suppression of Shmt2 or Nmdmc enhances lethality in parkin mutants (asterisks, χ2 two-tailed, 95% confidence intervals). (i) RNAi-mediated suppression of Shmt2 or Nmdmc in pink1 mutants. Expression levels were measured by real-time qPCR (mean±S.D.). The significant values relative to the control are indicated (asterisks, two-tailed unpaired t-test compared with Control). (j) RNAi-mediated suppression of Shmt2 or Nmdmc enhances lethality in pink1 mutants (asterisks, χ2 two-tailed, 95% confidence intervals). (k) Enhancement of the thoracic defects of pink1 mutants by RNAi-mediated suppression of Shmt2 or Nmdmc (asterisks, χ2 two-tailed, 95% confidence intervals). (l) Enhancement of the motor impairment of pink1 mutants by RNAi-mediated suppression of Nmdmc. Flies were tested using a standard climbing assay (mean±S.D.; asterisks, one-way ANOVA with Bonferroni's multiple comparison test). Genotypes in (a and b and d–l): Control: daGAL4; park25: park25, daGAL4/park25; pink1B9: pink1B9, daGAL4. All RNAi lines were driven by daGAL4. (c): Control: w1118
Mentions: To confirm that transcripts for the one-carbon metabolism genes Shmt2 and Nmdmc are induced in pink1 and parkin mutants as a compensatory mechanism for mitochondrial dysfunction, we tested the consequences of their suppression in these mutants. As ATF4 is required for the upregulation of SHMT2 and NMDMC in mammalian cells, we first determined the in vivo consequences of suppressing its expression in pink1 or parkin mutant flies. The knockdown of dAtf4 led to 11% and 84% lethality, respectively, in pink1 and parkin mutants (Figure 5a). Analysis of the surviving pink1 and parkin mutant adults with dAtf4 knockdown confirmed the downregulation of the dAtf4 transcript (Figure 5b) and revealed an increased penetrance of the crushed-thorax phenotype (Figures 5c and d). In addition, dAtf4 knockdown caused a decline in the climbing performance of pink1 mutants (Figure 5e). We next assessed the effect of dAtf4 knockdown on the levels of Shmt2 and Nmdmc in pink1 and parkin mutants. This caused a moderate decrease in Shmt2 and a more pronounced decrease in Nmdmc transcript levels (Figures 5f and g). Taken together, these results show that dAtf4 is involved in the upregulation of Shmt2 and Nmdmc in pink1 and parkin mutants. Next, we tested the effects of downregulating Shmt2 or Nmdmc in pink1 or parkin mutants. The knockdown of Shmt2 or Nmdmc led to 100% and 99% lethality, respectively, in parkin mutants (Figure 5h). In pink1 mutants, the knockdown of Shmt2 or Nmdmc reduced their respective transcript levels (Figure 5i), and caused 84 and 19% lethality, respectively (Figure 5j). Analysis of the surviving adults revealed an increased penetrance of the crushed-thorax phenotype in both Shmt2 and Nmdmc knockdown (Figure 5k). Furthermore, Nmdmc knockdown exhibited a dramatic decline in the climbing performance of pink1 mutants (Figure 5l). These results suggest that the one-carbon metabolism genes, Shmt2 and Nmdmc, were upregulated as a protective mechanism in pink1 and parkin mutants.

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