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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.


In silico identification of ATF4 as a regulator of transcriptional changes in pink1 and parkin mutants. (a) Enhanced expression of one-carbon metabolism genes Shmt2, Nmdmc, CG3999 (GCS P protein, glycine dehydrogenase) and CG6415 (GCS T protein, aminomethyl transferase) in heads of 3-day-old pink1 or parkin mutant flies. Relative levels to the w1118 control flies are indicated. Red corresponds to transcripts that are upregulated to a significant (P⩽0.05) level. Significance was determined using a two-tailed unpaired t-test (3 to 8 biological replicates per sample). (b) Workflow employed for the identification of upstream regulators of transcriptional changes upon loss of pink1 or parkin. (c) Analysis of upstream modulators of gene expression changes observed in pink1 (pink1B9) and parkin (park25) mutant flies. The term ‘young' corresponds to heads from 3-day-old pink1 and parkin mutant flies and ‘aged' to heads from 21- and 30-day-old parkin and pink1 mutant flies, respectively. The activation z-scores for ATF4 and TRIB3 are indicated. Red and blue correspond to significant positive (z ⩾2) and negative (z ⩽ −2) scores, respectively. Target molecules labelled in red correspond to positive transcripts regulated in the analysed data sets. (d) Changes in amino-acid abundance upon the loss of pink1 or parkin function. Relative levels to the w1118 control flies are indicated. Red and blue correspond to metabolites that are upregulated and downregulated to a significant level, respectively. ND corresponds to an amino-acid below detection threshold. Significance was determined using Welch's two-sample t-test (n=8). (e) Analysis of dAtf4 protein levels. Whole-fly lysates were analysed by western blotting using the indicated antibodies. The first two lanes correspond to a control for the specificity of the anti-dAtf4 antibody performed by analysing the levels of overexpressed dAtf4 in transgenic flies (da > UAS dAtf4). Genotypes: da>+, daGal4 > + da > UASdAtf4, daGal4 > UASdAtf4; control, w1118
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fig1: In silico identification of ATF4 as a regulator of transcriptional changes in pink1 and parkin mutants. (a) Enhanced expression of one-carbon metabolism genes Shmt2, Nmdmc, CG3999 (GCS P protein, glycine dehydrogenase) and CG6415 (GCS T protein, aminomethyl transferase) in heads of 3-day-old pink1 or parkin mutant flies. Relative levels to the w1118 control flies are indicated. Red corresponds to transcripts that are upregulated to a significant (P⩽0.05) level. Significance was determined using a two-tailed unpaired t-test (3 to 8 biological replicates per sample). (b) Workflow employed for the identification of upstream regulators of transcriptional changes upon loss of pink1 or parkin. (c) Analysis of upstream modulators of gene expression changes observed in pink1 (pink1B9) and parkin (park25) mutant flies. The term ‘young' corresponds to heads from 3-day-old pink1 and parkin mutant flies and ‘aged' to heads from 21- and 30-day-old parkin and pink1 mutant flies, respectively. The activation z-scores for ATF4 and TRIB3 are indicated. Red and blue correspond to significant positive (z ⩾2) and negative (z ⩽ −2) scores, respectively. Target molecules labelled in red correspond to positive transcripts regulated in the analysed data sets. (d) Changes in amino-acid abundance upon the loss of pink1 or parkin function. Relative levels to the w1118 control flies are indicated. Red and blue correspond to metabolites that are upregulated and downregulated to a significant level, respectively. ND corresponds to an amino-acid below detection threshold. Significance was determined using Welch's two-sample t-test (n=8). (e) Analysis of dAtf4 protein levels. Whole-fly lysates were analysed by western blotting using the indicated antibodies. The first two lanes correspond to a control for the specificity of the anti-dAtf4 antibody performed by analysing the levels of overexpressed dAtf4 in transgenic flies (da > UAS dAtf4). Genotypes: da>+, daGal4 > + da > UASdAtf4, daGal4 > UASdAtf4; control, w1118

Mentions: We have previously observed an upregulation of nucleotide metabolism pathways, including the one-carbon metabolism enzymes, in the heads of Drosophila pink1 (pink1B9) mutants flies.5 As Parkin cooperates with Pink1 in a pathway involved in mitochondrial quality control (QC), we compared the expression of genes involved in the mitochondrial one-carbon metabolism between pink1 and parkin (park25) mutants. This revealed a significant increase in mitochondrial transcripts for one-carbon enzymes in the heads of both pink1 and parkin mutants (Figure 1a), indicating that these transcripts are upregulated upon dysfunction of the Pink1/Parkin mitochondrial QC pathway.


dATF4 regulation of mitochondrial folate-mediated one-carbon metabolism is neuroprotective
In silico identification of ATF4 as a regulator of transcriptional changes in pink1 and parkin mutants. (a) Enhanced expression of one-carbon metabolism genes Shmt2, Nmdmc, CG3999 (GCS P protein, glycine dehydrogenase) and CG6415 (GCS T protein, aminomethyl transferase) in heads of 3-day-old pink1 or parkin mutant flies. Relative levels to the w1118 control flies are indicated. Red corresponds to transcripts that are upregulated to a significant (P⩽0.05) level. Significance was determined using a two-tailed unpaired t-test (3 to 8 biological replicates per sample). (b) Workflow employed for the identification of upstream regulators of transcriptional changes upon loss of pink1 or parkin. (c) Analysis of upstream modulators of gene expression changes observed in pink1 (pink1B9) and parkin (park25) mutant flies. The term ‘young' corresponds to heads from 3-day-old pink1 and parkin mutant flies and ‘aged' to heads from 21- and 30-day-old parkin and pink1 mutant flies, respectively. The activation z-scores for ATF4 and TRIB3 are indicated. Red and blue correspond to significant positive (z ⩾2) and negative (z ⩽ −2) scores, respectively. Target molecules labelled in red correspond to positive transcripts regulated in the analysed data sets. (d) Changes in amino-acid abundance upon the loss of pink1 or parkin function. Relative levels to the w1118 control flies are indicated. Red and blue correspond to metabolites that are upregulated and downregulated to a significant level, respectively. ND corresponds to an amino-acid below detection threshold. Significance was determined using Welch's two-sample t-test (n=8). (e) Analysis of dAtf4 protein levels. Whole-fly lysates were analysed by western blotting using the indicated antibodies. The first two lanes correspond to a control for the specificity of the anti-dAtf4 antibody performed by analysing the levels of overexpressed dAtf4 in transgenic flies (da > UAS dAtf4). Genotypes: da>+, daGal4 > + da > UASdAtf4, daGal4 > UASdAtf4; control, w1118
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fig1: In silico identification of ATF4 as a regulator of transcriptional changes in pink1 and parkin mutants. (a) Enhanced expression of one-carbon metabolism genes Shmt2, Nmdmc, CG3999 (GCS P protein, glycine dehydrogenase) and CG6415 (GCS T protein, aminomethyl transferase) in heads of 3-day-old pink1 or parkin mutant flies. Relative levels to the w1118 control flies are indicated. Red corresponds to transcripts that are upregulated to a significant (P⩽0.05) level. Significance was determined using a two-tailed unpaired t-test (3 to 8 biological replicates per sample). (b) Workflow employed for the identification of upstream regulators of transcriptional changes upon loss of pink1 or parkin. (c) Analysis of upstream modulators of gene expression changes observed in pink1 (pink1B9) and parkin (park25) mutant flies. The term ‘young' corresponds to heads from 3-day-old pink1 and parkin mutant flies and ‘aged' to heads from 21- and 30-day-old parkin and pink1 mutant flies, respectively. The activation z-scores for ATF4 and TRIB3 are indicated. Red and blue correspond to significant positive (z ⩾2) and negative (z ⩽ −2) scores, respectively. Target molecules labelled in red correspond to positive transcripts regulated in the analysed data sets. (d) Changes in amino-acid abundance upon the loss of pink1 or parkin function. Relative levels to the w1118 control flies are indicated. Red and blue correspond to metabolites that are upregulated and downregulated to a significant level, respectively. ND corresponds to an amino-acid below detection threshold. Significance was determined using Welch's two-sample t-test (n=8). (e) Analysis of dAtf4 protein levels. Whole-fly lysates were analysed by western blotting using the indicated antibodies. The first two lanes correspond to a control for the specificity of the anti-dAtf4 antibody performed by analysing the levels of overexpressed dAtf4 in transgenic flies (da > UAS dAtf4). Genotypes: da>+, daGal4 > + da > UASdAtf4, daGal4 > UASdAtf4; control, w1118
Mentions: We have previously observed an upregulation of nucleotide metabolism pathways, including the one-carbon metabolism enzymes, in the heads of Drosophila pink1 (pink1B9) mutants flies.5 As Parkin cooperates with Pink1 in a pathway involved in mitochondrial quality control (QC), we compared the expression of genes involved in the mitochondrial one-carbon metabolism between pink1 and parkin (park25) mutants. This revealed a significant increase in mitochondrial transcripts for one-carbon enzymes in the heads of both pink1 and parkin mutants (Figure 1a), indicating that these transcripts are upregulated upon dysfunction of the Pink1/Parkin mitochondrial QC pathway.

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.