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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

Neuronal loss in pink1 and parkin mutants is complemented by Shmt2 or Nmdmc. (a) Analysis of Shmt2 and Nmdmc expression levels (mean±S.D.; asterisks, two-tailed unpaired t-test relative to control). (b) Analysis of Shmt2 and Nmdmc protein levels. Whole-fly lysates were analysed using western blotting with the indicated antibodies. The 3-day-old flies were used for analysis of transcript and protein levels. (c) Expression of Shmt2 or Nmdmc rescues the loss of Δψm (mean±S.D.; asterisks, one-way ANOVA with Dunnett's multiple comparison test). (d) A whole mounted control fly brain showing TH-positive neurons, arrows indicate individual PPL1 cluster neurons. (e) Schematic diagram of a fly brain in sagittal orientation indicating the PPL1 cluster of dopaminergic neurons in yellow. (f) Expression of Shmt2 or Nmdmc rescues the loss of dopaminergic neurons in the PPL1 cluster of pink1 and parkin mutant flies (mean±S.D.; asterisks, one-way ANOVA with Dunnett's multiple comparison test). (g) RNAi-mediated suppression of dGcn2 fails to rescue the loss of dopaminergic neurons in the PPL1 cluster of pink1 or parkin mutant flies (mean±S.D.; asterisks, one-way ANOVA with Dunnett's multiple comparison test). Genotypes in (a and b): Control: daGAL4, Shmt2 and Nmdmc transgenes were driven by daGAL4, (c–g): Control: elavGAL4, Shmt2 and Nmdmc transgenes and RNAi dGcn2 were driven by elavGAL4
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fig6: Neuronal loss in pink1 and parkin mutants is complemented by Shmt2 or Nmdmc. (a) Analysis of Shmt2 and Nmdmc expression levels (mean±S.D.; asterisks, two-tailed unpaired t-test relative to control). (b) Analysis of Shmt2 and Nmdmc protein levels. Whole-fly lysates were analysed using western blotting with the indicated antibodies. The 3-day-old flies were used for analysis of transcript and protein levels. (c) Expression of Shmt2 or Nmdmc rescues the loss of Δψm (mean±S.D.; asterisks, one-way ANOVA with Dunnett's multiple comparison test). (d) A whole mounted control fly brain showing TH-positive neurons, arrows indicate individual PPL1 cluster neurons. (e) Schematic diagram of a fly brain in sagittal orientation indicating the PPL1 cluster of dopaminergic neurons in yellow. (f) Expression of Shmt2 or Nmdmc rescues the loss of dopaminergic neurons in the PPL1 cluster of pink1 and parkin mutant flies (mean±S.D.; asterisks, one-way ANOVA with Dunnett's multiple comparison test). (g) RNAi-mediated suppression of dGcn2 fails to rescue the loss of dopaminergic neurons in the PPL1 cluster of pink1 or parkin mutant flies (mean±S.D.; asterisks, one-way ANOVA with Dunnett's multiple comparison test). Genotypes in (a and b): Control: daGAL4, Shmt2 and Nmdmc transgenes were driven by daGAL4, (c–g): Control: elavGAL4, Shmt2 and Nmdmc transgenes and RNAi dGcn2 were driven by elavGAL4

Mentions: We previously showed that the pharmacological enhancement of folic acid metabolism confers a protective effect on the mitochondria of pink1 or parkin mutants.5 We therefore examined the effects of overexpressing one-carbon metabolism genes, Shmt2 and Nmdmc, in pink1 or parkin mutants. We first confirmed the overexpression of either Shmt2 or Nmdmc (Figures 6a and b) in adult flies. Next, by targeting the expression of these genes, using a neuronal driver, we rescued the mitochondrial function in neurons (Figure 6c) and the loss of dopaminergic neurons in the PPL1 cluster of both pink1 and parkin mutants (Figures 6d–f). Taken together, these results show that the one-carbon metabolism genes have a neuroprotective role in the Pink1/Parkin PD model. We showed that Shmt2 and Nmdmc expression is in part mediated by ATF4 in pink1 and parkin mutants (Figures 5f and g). ATF4 can be activated by the ER stress-inducible kinase PERK and Gcn2, a kinase that acts as a sensor of amino-acid depletion by binding uncharged tRNAs and phosphorylating eIF2α on serine 51 (reviewed in Kilberg et al.1). We showed that suppression of protein kinase R-like endoplasmic reticulum kinase (PERK) is neuroprotective in pink1 and parkin mutants.16 As we observed significant alterations in amino-acid levels in pink1 and parkin mutants (Figure 1d), we tested whether Drosophila Gcn2 (dGcn2) also plays a role in the activation of ATF4 in these mutants. RNAi-mediated suppression of dGcn2 failed to rescue the neuronal loss in pink1 or parkin mutant flies (Figure 6g). Together, these results indicate that in pink1 and parkin mutants, ATF4 is regulated via PERK.


dATF4 regulation of mitochondrial folate-mediated one-carbon metabolism is neuroprotective
Neuronal loss in pink1 and parkin mutants is complemented by Shmt2 or Nmdmc. (a) Analysis of Shmt2 and Nmdmc expression levels (mean±S.D.; asterisks, two-tailed unpaired t-test relative to control). (b) Analysis of Shmt2 and Nmdmc protein levels. Whole-fly lysates were analysed using western blotting with the indicated antibodies. The 3-day-old flies were used for analysis of transcript and protein levels. (c) Expression of Shmt2 or Nmdmc rescues the loss of Δψm (mean±S.D.; asterisks, one-way ANOVA with Dunnett's multiple comparison test). (d) A whole mounted control fly brain showing TH-positive neurons, arrows indicate individual PPL1 cluster neurons. (e) Schematic diagram of a fly brain in sagittal orientation indicating the PPL1 cluster of dopaminergic neurons in yellow. (f) Expression of Shmt2 or Nmdmc rescues the loss of dopaminergic neurons in the PPL1 cluster of pink1 and parkin mutant flies (mean±S.D.; asterisks, one-way ANOVA with Dunnett's multiple comparison test). (g) RNAi-mediated suppression of dGcn2 fails to rescue the loss of dopaminergic neurons in the PPL1 cluster of pink1 or parkin mutant flies (mean±S.D.; asterisks, one-way ANOVA with Dunnett's multiple comparison test). Genotypes in (a and b): Control: daGAL4, Shmt2 and Nmdmc transgenes were driven by daGAL4, (c–g): Control: elavGAL4, Shmt2 and Nmdmc transgenes and RNAi dGcn2 were driven by elavGAL4
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5384021&req=5

fig6: Neuronal loss in pink1 and parkin mutants is complemented by Shmt2 or Nmdmc. (a) Analysis of Shmt2 and Nmdmc expression levels (mean±S.D.; asterisks, two-tailed unpaired t-test relative to control). (b) Analysis of Shmt2 and Nmdmc protein levels. Whole-fly lysates were analysed using western blotting with the indicated antibodies. The 3-day-old flies were used for analysis of transcript and protein levels. (c) Expression of Shmt2 or Nmdmc rescues the loss of Δψm (mean±S.D.; asterisks, one-way ANOVA with Dunnett's multiple comparison test). (d) A whole mounted control fly brain showing TH-positive neurons, arrows indicate individual PPL1 cluster neurons. (e) Schematic diagram of a fly brain in sagittal orientation indicating the PPL1 cluster of dopaminergic neurons in yellow. (f) Expression of Shmt2 or Nmdmc rescues the loss of dopaminergic neurons in the PPL1 cluster of pink1 and parkin mutant flies (mean±S.D.; asterisks, one-way ANOVA with Dunnett's multiple comparison test). (g) RNAi-mediated suppression of dGcn2 fails to rescue the loss of dopaminergic neurons in the PPL1 cluster of pink1 or parkin mutant flies (mean±S.D.; asterisks, one-way ANOVA with Dunnett's multiple comparison test). Genotypes in (a and b): Control: daGAL4, Shmt2 and Nmdmc transgenes were driven by daGAL4, (c–g): Control: elavGAL4, Shmt2 and Nmdmc transgenes and RNAi dGcn2 were driven by elavGAL4
Mentions: We previously showed that the pharmacological enhancement of folic acid metabolism confers a protective effect on the mitochondria of pink1 or parkin mutants.5 We therefore examined the effects of overexpressing one-carbon metabolism genes, Shmt2 and Nmdmc, in pink1 or parkin mutants. We first confirmed the overexpression of either Shmt2 or Nmdmc (Figures 6a and b) in adult flies. Next, by targeting the expression of these genes, using a neuronal driver, we rescued the mitochondrial function in neurons (Figure 6c) and the loss of dopaminergic neurons in the PPL1 cluster of both pink1 and parkin mutants (Figures 6d–f). Taken together, these results show that the one-carbon metabolism genes have a neuroprotective role in the Pink1/Parkin PD model. We showed that Shmt2 and Nmdmc expression is in part mediated by ATF4 in pink1 and parkin mutants (Figures 5f and g). ATF4 can be activated by the ER stress-inducible kinase PERK and Gcn2, a kinase that acts as a sensor of amino-acid depletion by binding uncharged tRNAs and phosphorylating eIF2α on serine 51 (reviewed in Kilberg et al.1). We showed that suppression of protein kinase R-like endoplasmic reticulum kinase (PERK) is neuroprotective in pink1 and parkin mutants.16 As we observed significant alterations in amino-acid levels in pink1 and parkin mutants (Figure 1d), we tested whether Drosophila Gcn2 (dGcn2) also plays a role in the activation of ATF4 in these mutants. RNAi-mediated suppression of dGcn2 failed to rescue the neuronal loss in pink1 or parkin mutant flies (Figure 6g). Together, these results indicate that in pink1 and parkin mutants, ATF4 is regulated via PERK.

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