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Enhancing nucleotide metabolism protects against mitochondrial dysfunction and neurodegeneration in a PINK1 model of Parkinson’s disease

Abstract

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.

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Figure 1: Loss of Pink1 results in a metabolic stress response.
Figure 2: dNK enhances mitochondrial function by promoting organellar biogenesis.
Figure 3: Mitochondrial dysfunction in Drosophila pink1 mutants is complemented by dNK.
Figure 4: Targeted neuronal expression of dNK rescues mitochondrial dysfunction in pink1 mutants.
Figure 5: Targeted neuronal expression of dNK rescues neurodegeneration in pink1 mutants.
Figure 6: Dietary supplementation with dNs or FA enhances mitochondrial function and biogenesis in pink1 mutants.
Figure 7: Dietary supplementation with dNs or FA suppresses pink1 mutant phenotypes.
Figure 8: An exogenous supply of dNs and FA suppresses mitochondrial dysfunction on loss of PINK1 in human neuroblastoma cells.

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Acknowledgements

We would like to thank J. Silber (Institut Jacques Monod, France), A. Whitworth (University of Sheffield, UK), T. Rival (Aix-Marseille Université, France), the Vienna Drosophila RNAi Center and the Bloomington Drosophila Stock Center for fly stocks; and J. Parmar for fly food preparation. We thank D. Green for comments on the manuscript and helpful discussions. We thank A. Antonov for advice on advanced biostatistics. We also thank M. Locker for helpful discussions.

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Authors and Affiliations

Authors

Contributions

A.Y.A., G.R.M., L.M.M., R.T. and S.H.Y.L. conceived and designed the experiments. A.Y.A., D.D., I.P.d.C., L.M.M., P.R.A., R.T., S.G., S.L. and S.H.Y.L. performed the experiments and analysed the data. R.T. did most of the experimental work and analysis. E.D. contributed materials. A.E.W., H.P-F. and P.N. provided experimental and conceptual advice. G.R.M., L.M.M., R.T., S.G. and S.H.Y.L wrote the paper. S.H.Y.L. and L.M.M. contributed equally as joint last authors.

Corresponding authors

Correspondence to Samantha H. Y. Loh or L. Miguel Martins.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Loss of Pink1 results in a metabolic stress response.

RNA and metabolites were isolated from 3-day-old control and pink1 male flies. (a) Quantitative RT–PCR validation for a subset of genes found to be upregulated in the microarray analysis of Drosophila pink1 mutants and that encode for metabolic enzymes. Circled numbers are related to the metabolic reactions shown in b. Data are shown as the mean±SEM, n values are indicated in the bars, asterisks, two-tailed unpaired t test. See also Supplementary Fig. 1 and Supplementary Table 1–3 and 9 for statistics source data. (b) Coordinated changes in metabolite abundance upon loss of pink1 function. On the left, the relative levels of selected metabolites in pink1 mutant flies, detected through GC-MS and LC-MS/MS analysis. A schematic diagram is shown in which these metabolites are depicted with respect to the selected upregulated transcripts. Orange and cyan correspond to metabolites that are respectively upregulated and downregulated to a significant level. The statistical significance was determined using Welch’s Two Sample t-test (n = 8). See also Supplementary Fig. 1 and Supplementary Table 4 and 5.

Supplementary Figure 2 dNK enhances mitochondrial function by promoting organellar biogenesis.

(a) Enhanced mtDNA synthesis in dNK transgenic flies. DNA synthesis was assessed using a BrdU assay (mean±SEM, n = 12, asterisks, two-tailed paired t test). (b) dNK flies show an increase in mtDNA. The ratio of mtDNA to nDNA was measured by real-time PCR using third instar larvae and 2-day-old flies with the indicated genotypes (mean±SD, n = 9, asterisks, two-tailed paired t test). (c) dNK flies show an increase in mitochondrial OXPHOS proteins. Immunoblot of samples prepared from whole 2-day-old males. α-tubulin, loading control. (d) Enhanced respiration in dNK flies. Data are shown as the mean±SD (n = 3 per genotype, asterisks, two-tailed unpaired t test). (e) dNK flies show a transcriptional upregulation of PGC-1 family homologue Spargel and the nuclear-encoded mtDNA binding proteins Tfam, mtTFB1 and mtTFB2. Data are shown as the mean±SEM (n values are indicated in the bars, asterisks, two-tailed unpaired t test). (f) dNK expression increases protein levels of mtTFA. Lysates prepared from adult flies were subjected to western blot analysis with the indicated antibodies. (g) RNAi-mediated suppression of Tfam. Expression levels were measured by real-time PCR (relative mean Ct±SEM, n values are indicated). Statistically significant values relative to the control are indicated (one-way ANOVA with Bonferroni’s multiple comparison test). (h) Tfam is required for the dNK-mediated increase in mtDNA. The ratio of mtDNA to nDNA was measured by real-time PCR using 2-day-old flies with the indicated genotypes (mean±SEM, n values are indicated, asterisks, one-way ANOVA with Bonferroni’s multiple comparison test, ***p<0.0001). (i) dNK expression results in a generalised ATP increase in both young (2-day-old) and old (40-day-old) flies. Data are shown as the mean±SD from three independent experiments (n = 3 per genotype, asterisks, two-tailed paired t test). (j) Ubiquitous expression of dNK enhances locomotor activity. 16 flies were tested for each genotype. (k) Ubiquitous expression of dNK enhances climbing ability. Flies were tested using a standard climbing assay (mean±SEM, n = 100 flies per genotype, asterisks, two-tailed unpaired t test). See also Supplementary Figs 1 and 9 and Supplementary Table 9 and for statistics source data of (d), (e), (g), (h) and (i).

Supplementary Figure 3 Mitochondrial dysfunction in Drosophila pink1 mutants is complemented by dNK.

(a) Expression of dNK partially reverses metabolic shifts in pink1 mutants. Orange and cyan correspond to metabolites that are respectively upregulated and downregulated to a significant level. The statistical significance was determined using Welch’s Two Sample t-test (n = 8). See also Supplementary Table 6. (b) dNK expression increases mtDNA levels in pink1 mutants. The ratio of mtDNA to nDNA was measured by real-time PCR in flies with the indicated genotypes (mean±SD, n = 6 per genotype, asterisks, one-way ANOVA with Bonferroni’s multiple comparison test). (c) Expression of dNK restores the levels of mitochondrial respiratory complexes in pink1 mutants. Whole-fly lysates were analysed by western blot analysis using the indicated antibodies. (d) dNK expression rescues ATP levels in pink1 mutants. Data are shown as the mean±SD (n = 6 per genotype, asterisks, one-way ANOVA with Bonferroni’s multiple comparison test). (e) dNK expression enhances respiration in pink1 mutants. Data are shown as the mean±SD (n = 3 per genotype, asterisks, two-tailed unpaired t test). See Supplementary Table 9 for statistics source data. (f) dNK expression suppresses motor impairment in pink1 mutants. Flies were tested using a standard climbing assay (mean±SEM, n = 100 flies per genotype, asterisks, one-way ANOVA with Dunnett’s multiple comparison test). (g) dNK expression suppresses flight muscle defects observed in pink1 mutants. dNK expression rescues mitochondrial defects in pink1 mutants (my, myofibrils; m, mitochondria; yellow outlines, mitochondria). See also Supplementary Figs 3 and 9.

Supplementary Figure 4 Targeted neuronal expression of dNK rescues mitochondrial dysfunction in pink1 mutants.

(a) Enhanced expression of components of both the de novo and salvage nucleotide synthesis pathways in adult heads of pink1 mutant flies. Data are shown as the mean±SEM (n values are indicated in the bars, asterisks, two-tailed unpaired t test). See Supplementary Table 9 for statistics source data. (b) Enhanced respiration in the heads of elav>dNK flies. Data are shown as the mean±SEM (n values are indicated, asterisks, two-tailed unpaired t test). (c) Neuronal expression of dNK enhances ATP levels. Data are shown as the mean±SEM (n = 6 per sample, asterisks, two-tailed unpaired t test). (d) Neuronal expression of dNK enhanced locomotor activity. Data are shown as the mean±SEM (n values are indicated, asterisks, two-tailed unpaired t test). (e) Neuronal expression of dNK enhanced climbing ability (mean±SEM, n = 100 flies per genotype, asterisks, two-tailed unpaired t test). See also Supplementary Fig. 4.

Supplementary Figure 5 Targeted neuronal expression of dNK rescues neurodegeneration in pink1 mutants.

(a) Neuronal expression of dNK restores the tyrosine hydroxylase (TH) levels in pink1 mutants. Fly head lysates were analysed using the indicated antibodies. (b) Expression of dNK rescues the loss of dopaminergic neurons in the PPL1 cluster of pink1 mutant flies. Data are shown as the mean ± SEM (n values are indicated, asterisks, one-way ANOVA with Bonferroni’s multiple comparison test). Datasets labelled ‘control’ and ‘pink1B9’ are also used in Figs. 6h and 6i. (c) Neuronal expression of dNK promotes an increase in neuronal mitochondrial mass of pink1 mutants. Mitochondrial mass was calculated as the ratio of co-localisation between the TMRM signal (mitochondria) and calcein blue (whole cells). Data are the mean±SEM (n = 6 per genotype, asterisks, two-tailed paired t test). Datasets labelled ‘control’ and ‘pink1B9’ are also used in Figs. 6d and 6e. (d) Neuronal expression of dNK reverses the loss of Δψm in pink1 mutants. The Δψm is represented as percentage of control. Data are shown as the mean±SEM (n = 7 per genotype, asterisks, two-tailed unpaired t test). Datasets labelled ‘control’ and ‘pink1B9’ are also used in Figs. 6f and 6g. (e) Neuronal expression of dNK enhances respiration in pink1 mutants. Data are shown as the mean±SD (n values are indicated, asterisks, two-tailed unpaired t test). Complex I and II, and complex IV were measured in coupled and uncoupled mitochondria, respectively. See Supplementary Table 9 for statistics source data. (f) Neuronal expression of dNK rescues the thoracic defects of pink1 mutants (n = 255 for pink1B9, n = 227 for pink1B9; elav>dNK, n = 187 for pink1B9; elav>pink1), asterisks, chi-square two-tailed, 95% confidence intervals). (g) Neuronal expression of dNK rescues the motor impairment of pink1 mutants. Mean ± SEM, n = 100 flies per genotype, asterisks, two-tailed paired t test, relative to pink1B9). See also Supplementary Fig. 5.

Supplementary Figure 6 Dietary supplementation with deoxyribonucleosides or folic acid enhances mitochondrial function and biogenesis in pink1 mutants.

(a) Dietary supplementation with deoxyribonucleosides (dNs, 0.5 mg/ml each dN) or folic acid (4 mM) promotes an upregulation of biochemical components of nucleotide metabolism pathways in pink1 mutants. Orange corresponds to metabolites that are upregulated to a significant level (p<0.05). Orange boxes with a dashed outline correspond to comparisons with lower statistical significance (0.05<p<0.10). Statistical significance was determined using Welch’s Two Sample t-test (n = 5). See also Supplementary Table 7. (b) and (c) Dietary supplementation with dNs (b) or FA (c) during the adult stage promotes an increase in mtDNA. The ratio of mtDNA to nDNA was measured by real-time PCR using 2-day-old flies with the indicated genotypes (mean±SD, n = 9 (b), n = 3 (c) per genotype). Statistically significant values are indicated by asterisks (one-way ANOVA with Bonferroni’s multiple comparison test). (d) and (e) dNs or FA promote an increase in neuronal mitochondrial mass of pink1 mutants. Data are shown as the mean±SEM (n = 6 for dNs, n = 7 for FA). Statistical significance is indicated by asterisks (two-tailed paired t test). Datasets labelled ‘control’ and ‘pink1B9’ are also used in Fig. 5c. (f) and (g) dNs or FA reverse the loss of Δψm in pink1 mutants. The Δψm is represented as percentage of control. The error bars represent the mean±SEM (n = 7). Statistical significance is indicated by asterisks (two-tailed unpaired t test). Datasets labelled ‘control’ and ‘pink1B9’ are also used in Fig. 5d. (h) and (i) dNs or FA reverse the loss of dopaminergic neurons in the PPL1 cluster of pink1 mutant. Data are shown as the mean±SEM (n values are indicated in the bars, one-way ANOVA with Bonferroni’s multiple comparison test). Datasets labelled ‘control’ and ‘pink1B9’ are also used in Fig. 5b. (j) and (k) dNs or FA promote the transcriptional upregulation of the nuclear-encoded mtDNA binding proteins Tfam, mtTFB1 and mtTFB2 in pink1 mutants. The error bars represent SEM values (n values are indicated in the bars), and the asterisks indicate statistically significant values (two-tailed unpaired t test) relative to pink1 flies on a normal diet. See also Supplementary Fig. 6 and Supplementary Table 9 for statistics source data of (c), (j) and (k).

Supplementary Figure 7 Dietary supplementation with deoxyribonucleosides or folic acid suppresses pink1 mutant phenotypes.

(a) Suppression of flight muscle degeneration and mitochondrial defects in pink1 mutants by dNs or FA. Ultrastructural analysis of the indirect flight muscles from pink1 mutant flies raised on dNs or FA-supplemented food (my, myofibrils; m, mitochondria; yellow outlines, mitochondria). (b) Dietary supplementation with dNs or FA rescues the thoracic defects of pink1 mutants. pink1 mutants were exposed to a dNs or FA-supplemented diet after egg laying. P-values are indicated (chi-square two-tailed, 95% confidence intervals, n = 454 for normal diet, n = 448 for +dNs and n = 669 for +FA). (c) Dietary supplementation with dNs or FA rescues the flight defects of pink1 mutants. Data are shown as mean±SEM (n = 150 flies per condition). Statistically significant values relative to pink1B9 are indicated by asterisks (one-way ANOVA with Bonferroni’s multiple comparison test). Datasets labelled ‘control’ and ‘pink1B9’ are also used in Supplementary Fig. 4c. (d) and (e) Dietary supplementation with dNs or FA during adult stage rescues the motor impairment of pink1 mutants. pink1 mutants were exposed to a dNs or FA supplemented diet after eclosion. Flies were tested using a standard climbing assay (mean±SEM, n = 100 flies per condition). Statistical significance is indicated (two-tailed unpaired t test). See also Supplementary Fig. 7.

Supplementary Figure 8 An exogenous supply of deoxyribonucleosides and folic acid suppresses mitochondrial dysfunction upon loss of PINK1 in human neuroblastoma cells.

(a) Genes encoding for enzymes of the purine biosynthetic and nucleotide salvage pathways are significantly upregulated in a subset of PD patients with PINK1 mutations. Delta Ct values (ΔCt) for each of the analysed transcripts were normalised to GAPDH. Means are represented by horizontal bars. P-values (two-tailed unpaired t test) relative to controls are indicated. See Supplementary Table 9 for statistics source data. (b) An exogenous supply of dNs (i) or folate (ii) reversed the reduction in the basal mitochondrial membrane potential in PINK1 KD cells. Cells were grown for 24 hr in media supplemented with dNs (20 μM each dN) or FA (300 μM) and were compared to cells grown in normal media. (i) mean±SEM, n = 240 cells of three separate clones, data from 6 independent experiments. (ii) mean±SEM, n = 180 cells of three separate clones, data from 6 independent experiments. Statistical significance is indicated by asterisks (two-tailed paired t test). (c) The reversal of oligomycin-induced Δψm by dNs and FA. In PINK1 KD cells (Ci), oligomycin caused marked mitochondrial depolarisation, whereas the exogenous addition of either dNs (Cii) or FA (Ciii) completely blocked depolarisation. Cells were grown for 24 hr in media supplemented with dNs (20 μM each dN) or FA (300 μM) and were compared to cells grown in normal media. Error bars represent mean±SEM (n = 180 cells). (d, e) Reversal of the loss of NADH redox state in PINK1 KD cells by dNs and FA. The resting level of NADH autofluorescence was measured by determining the ratio between the signal for the maximally oxidised condition (response to 1 μM FCCP) and the signal for the maximally reduced condition (response to 1 mM NaCN) to determine the baseline ‘redox state’ in control cells (di). PINK1 KD cells showed a decreased redox state (dii and e), whereas the exogenous supply of either dNs or FA recovered the redox state (diii, div and e). Cells were grown for 24 hr in media supplemented with dNs (20 μM each dN) or FA (300 μM) and were compared to cells grown in normal media. The error bars represent the mean±SEM (n = 180 cells). Statistical significance is indicated by asterisks (two-tailed paired t test).

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Tufi, R., Gandhi, S., de Castro, I. et al. Enhancing nucleotide metabolism protects against mitochondrial dysfunction and neurodegeneration in a PINK1 model of Parkinson’s disease. Nat Cell Biol 16, 157–166 (2014). https://doi.org/10.1038/ncb2901

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