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Enhancing Nucleotide Metabolism Protects Against Mitochondrial Dysfunction and Neurodegeneration in a PINK1 Model of Parkinson’S Disease

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Enhancing Nucleotide Metabolism Protects Against Mitochondrial Dysfunction and Neurodegeneration in a PINK1 Model of Parkinson’S Disease ARTICLES Enhancing nucleotide metabolism protects against mitochondrial dysfunction and neurodegeneration in a PINK1 model of Parkinson's disease Roberta Tufi1, Sonia Gandhi2, Inês P. de Castro1, Susann Lehmann1, Plamena R. Angelova2, David Dinsdale1, Emma Deas2, Hélène Plun-Favreau2, Pierluigi Nicotera3, Andrey Y. Abramov2, Anne E. Willis1, Giovanna R. Mallucci1, Samantha H. Y. Loh1,4 and L. Miguel Martins1,4 Mutations in PINK1 cause early-onset Parkinson's disease (PD). Studies in Drosophila melanogaster have highlighted mitochondrial dysfunction on loss of Pink1 as a central mechanism of PD pathogenesis. Here we show that global analysis of transcriptional changes in Drosophila pink1 mutants reveals an upregulation of genes involved in nucleotide metabolism, critical for neuronal mitochondrial DNA synthesis. These key transcriptional changes were also detected in brains of PD patients harbouring PINK1 mutations. We demonstrate that genetic enhancement of the nucleotide salvage pathway in neurons of pink1 mutant flies rescues mitochondrial impairment. In addition, pharmacological approaches enhancing nucleotide pools reduce mitochondrial dysfunction caused by Pink1 deficiency. We conclude that loss of Pink1 evokes the activation of a previously unidentified metabolic reprogramming pathway to increase nucleotide pools and promote mitochondrial biogenesis. We propose that targeting strategies enhancing nucleotide synthesis pathways may reverse mitochondrial dysfunction and rescue neurodegeneration in PD and, potentially, other diseases linked to mitochondrial impairment. The role of mitochondrial impairment in PD has long been debated. By combining transcriptional and metabolic profiling, we have Recently, the identification of causative mutations in PINK1, a gene uncovered significant alterations in the nucleotide metabolism encoding a mitochondrial kinase in PD patients has renewed interest in networks of pink1 mutant flies. In the cell, two metabolic pathways, the role of mitochondrial damage in PD (ref. 1). referred to as the de novo and salvage pathways, are involved Cells have evolved several lines of defence to cope with damaged in nucleotide metabolism. Postmitotic cells such as neurons are mitochondria2. Molecular quality control represents the first level of reported to lack the de novo biosynthetic pathways for nucleotide the mitochondrial defence mechanism. This involves the upregulation generation and instead rely on the salvage pathway6. The conversion of nuclear genes that encode for mitochondrial chaperones and of deoxyribonucleosides (dNs) to their monophosphate forms is proteases that remove misfolded and non-assembled polypeptides the rate-limiting step in the salvage pathway7, and is catalysed in mitochondria, a form of mitochondrial retrograde signalling by deoxyribonucleoside kinases (dNKs). Drosophila dNK is a that is commonly referred to as the mitochondrial unfolded highly efficient and multi-substrate single dNK acting on the protein response3 (UPRmt). nucleotide salvage pathway8. Here we show that both the genetic Invertebrates such as Drosophila have recently emerged as enhancement of the nucleotide salvage pathway by overexpression powerful model systems to study the mechanisms of PD-associated of dNK and the pharmacological manipulation of nucleotide neurodegeneration. These models are also excellent in vivo systems for metabolism enhance mitochondrial biogenesis, thus suppressing the testing of therapeutic compounds4. mitochondrial dysfunction associated with the PD phenotypes due Previously, we observed that Drosophila pink1 mutants exhibit to Pink1 deficiency. significant upregulation of key markers of the UPRmt (ref. 5). To extend Enhancement of nucleotide metabolism might therefore be of these observations, here we proceeded with an unbiased identification therapeutic benefit in human age-related neurological disorders linked of upregulated transcripts in pink1 mutant flies. to mitochondrial dysfunction. 1MRC Toxicology Unit, Lancaster Road, Leicester LE1 9HN, UK. 2Department of Molecular Neuroscience, Institute of Neurology, Queen Square, London WC1N 3BG, UK. 3German Centre for Neurodegenerative Diseases (DZNE), Ludwig-Erhard-Allee 2, 53175 Bonn, Germany. 4Correspondence should be addressed to S.H.Y.L. or L.M.M. (e-mail: [email protected] or [email protected]) Received 3 April 2013; accepted 29 November 2013; published online 19 January 2014; DOI: 10.1038/ncb2901 NATURE CELL BIOLOGY VOLUME 16j NUMBER 2j FEBRUARY 2014 157 © 2014 Macmillan Publishers Limited. All rights reserved. ARTICLES a Fold induction 0 1 2 3 45 Pathway 5 1 Shmt2/CG3011 Control 6 ** Pink1B9 5 Glycine and serine metabolism 2 CG3999 6 ** 3 3 CG6415 5 ** 5 4 Mthfd2/Nmdmc 6 * 1-carbon pool by folate 5 5 Dhfr 6 * 5 6 Paics/ade2 6 *** Purine biosynthesis 4 7 atic/CG11089 7 * 5 8 dNK Nucleotide salvage 6 * b Pink1B9 Control Mitochondrion Cytoplasm Glycine 1.34 Serine Serine Histidine Serine 1.65 5 THF THF 1 DHF Methionine 1.31 Glycine Glycine Glycine cleavage 2,3 1.73 5,10-methylene-THF Formimino- Methionine sulphoxide system 5,10-methylene-THF glutamate 4 Homocysteine 2.19 5,10-methenyl-THF 5,10-methenyl-THF 4 Deoxycytidine Histidine 1.36 10-formyl-THF 10-formyl-THF 8 Tryptophan 1.28 6,7 dCMP THF Kynurenine 1.66 Purines Formate Formate dCTP Pyrophosphate 2.30 Methionine Acetyl-CoA 5-methyl-THF dTMP UMP Glutamate 2.53 Oxaloacetate sulphoxide 8 Glutamine 1.17 Methionine Orotate Malate CoA Citrate 0.43 Acetyl-CoA Deoxyadenosine Citrate 0.31 Glutamine Homocysteine Deoxyguanosine Fumarate TCA cycle Isocitrate α-ketoglutarate 8.57 Kynurenine α Succinate 0.42 Succinate -ketoglutarate Glutamate Glutamate N-formylkynurenine Fumarate 0.75 Succinyl-CoA Malate 0.29 Tryptophan Deoxyguanosine 0.48 12Shmt2/CG3011 CG39993 CG64154 Mthfd2/Nmdmc56 Dhfr Paics/ade27 atic/CG110898 dNK Figure 1 Loss of Pink1 results in a metabolic stress response. RNA and metabolite abundance on loss of pink1 function. On the left, the relative metabolites were isolated from 3-day-old control and pink1 mutant male levels of selected metabolites in pink1 mutant flies, detected through gas flies. (a) Quantitative validation by PCR with reverse transcription for chromatography with mass spectrometry and liquid chromatography with a subset of genes found to be upregulated in the microarray analysis tandem mass spectrometry analysis. A schematic diagram is also shown, of Drosophila pink1 mutants and that encode for metabolic enzymes. in which these metabolites are depicted with respect to the selected Circled numbers are related to the metabolic reactions shown in b. Data upregulated transcripts. Orange and cyan correspond to metabolites that are shown as the mean ± s:e:m:; n values are indicated in the bars; are upregulated and downregulated, respectively, to a significant level. The asterisks, two-tailed unpaired t -test. See also Supplementary Fig. 1 and statistical significance was determined using Welch's two-sample t -test Tables 1–3 and 9 for statistics source data. (b) Coordinated changes in (n D 8). See also Supplementary Fig. 1 and Tables 4 and 5. RESULTS mitochondrial chaperones hsp-60 and hsc-70-5, markers of the UPRmt Identification of an altered metabolic signature in pink1 activation5. To determine the full complement of transcripts that are mutant flies upregulated in pink1 mutant flies, we employed microarray technology Previous studies have demonstrated that Drosophila pink1 mu- coupled with an in silico analysis approach (experimental outline, tants exhibit transcriptional upregulation of the nuclear-encoded Supplementary Fig. 1a) . Using the RankProducts method to identify 158 NATURE CELL BIOLOGY VOLUME 16j NUMBER 2j FEBRUARY 2014 © 2014 Macmillan Publishers Limited. All rights reserved. ARTICLES a b c d Control (da > +) ) 140 da > dNK 125 * –1 100 Controlda (da > >dNK +) M ml *** r (K) 120 –1 * 100 50 C-V-α 80 100 CIII-Core 2 80 75 37 CIV-1 60 60 50 40 40 ** mtDNA/nDNA 25 CII-SDHB 20 (OD per mg DNA) 25 BrdU incorporation BrdU 20 CI-NDUFB8 (percentage of control) (percentage flux per fly (pmol s 0 0 2 0 Larvae Adults α nDNA mtDNA 50 -tubulin O Complex I Complex II e f g h Control (da > +) 2.5 da > dNK 1.0 150 ** *** P = 0.09 2.0 0.8 * da > + da > dNK * * M 100 1.5 * r (K) Tfam 0.6 25 *** 1.0 α-tubulin 0.4 50 50 mtDNA/nDNA Fold induction Fold 0.5 0.2 3 6 6 6 6 7 3 3 of control) (percentage 0 0 0 Relative mRNA content (Tfam) n = 6)n = 4)n = 3) n = 3)n = 6)n = 4) n = 3) Tfam 2 ( Spargel no. no. 2 ( no. 2 (no. 2 ( mtTFB1 mtTFB2 Control ( Control ( da > dNK ( m-RNAi-RNAi -RNAim-RNAi da > Tfa da > Tfa da > dNK; Tfam da > dNK; Tfam i j k da > dNK Finish Control (da > +) 150 300 Infrared beam 25 * *** –1 125 20 100 200 15 Start ossing h ossing 75 ** 10 50 100 rage cr rage 5 Relative ATP levels Relative ATP 25 (s) latency Climbing Ave 0 0 0 240 0 60 120 180 240 Control Age (days) Time (h) (da >da +) > dNK Figure 2 dNK enhances mitochondrial function by promoting organellar suppression of Tfam. Expression levels were measured by real-time PCR biogenesis. (a) Enhanced mtDNA synthesis in dNK transgenic flies. DNA (relative mean Ct±s:e:m:, n values are indicated). Statistically significant synthesis was assessed using a BrdU assay (mean±s:e:m:; n D 12; asterisks, values relative to the control are indicated (one-way analysis of variance two-tailed paired t -test). (b) dNK flies show an increase in mtDNA. The (ANOVA) with Bonferroni's multiple comparison test). (h) Tfam is required ratio of mtDNA to nuclear DNA (nDNA) was measured by real-time PCR for the dNK-mediated increase in mtDNA. The ratio of mtDNA to nDNA using third-instar larvae and 2-day-old flies with the indicated genotypes was measured by real-time PCR using 2-day-old flies with the indicated (mean±s:d:; n D 9; asterisks, two-tailed paired t -test). (c) dNK flies show an genotypes (mean ± s:e:m:; n values are indicated; asterisks, one-way increase in mitochondrial oxidative phosphorylation proteins.
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