Tissue- and cell-specific mitochondrial defect in Parkin-deficient mice - PubMed (original) (raw)

. 2014 Jun 24;9(6):e99898.

doi: 10.1371/journal.pone.0099898. eCollection 2014.

Clément A Gautier 1, Anne-Laure Bulteau 2, Rosa Ferrando-Miguel 1, Caroline Gouarne 3, Marc Giraudon Paoli 3, Rebecca Pruss 3, Françoise Auchère 4, Caroline L'Hermitte-Stead 2, Frédéric Bouillaud 2, Alexis Brice 5, Olga Corti 1, Anne Lombès 2

Affiliations

Tissue- and cell-specific mitochondrial defect in Parkin-deficient mice

Maria Damiano et al. PLoS One. 2014.

Abstract

Loss of Parkin, encoded by PARK2 gene, is a major cause of autosomal recessive Parkinson's disease. In Drosophila and mammalian cell models Parkin has been shown in to play a role in various processes essential to maintenance of mitochondrial quality, including mitochondrial dynamics, biogenesis and degradation. However, the relevance of altered mitochondrial quality control mechanisms to neuronal survival in vivo is still under debate. We addressed this issue in the brain of PARK2-/- mice using an integrated mitochondrial evaluation, including analysis of respiration by polarography or by fluorescence, respiratory complexes activity by spectrophotometric assays, mitochondrial membrane potential by rhodamine 123 fluorescence, mitochondrial DNA content by real time PCR, and oxidative stress by total glutathione measurement, proteasome activity, SOD2 expression and proteins oxidative damage. Respiration rates were lowered in PARK2-/- brain with high resolution but not standard respirometry. This defect was specific to the striatum, where it was prominent in neurons but less severe in astrocytes. It was present in primary embryonic cells and did not worsen in vivo from 9 to 24 months of age. It was not associated with any respiratory complex defect, including complex I. Mitochondrial inner membrane potential in PARK2-/- mice was similar to that of wild-type mice but showed increased sensitivity to uncoupling with ageing in striatum. The presence of oxidative stress was suggested in the striatum by increased mitochondrial glutathione content and oxidative adducts but normal proteasome activity showed efficient compensation. SOD2 expression was increased only in the striatum of PARK2-/- mice at 24 months of age. Altogether our results show a tissue-specific mitochondrial defect, present early in life of PARK2-/- mice, mildly affecting respiration, without prominent impact on mitochondrial membrane potential, whose underlying mechanisms remain to be elucidated, as complex I defect and prominent oxidative damage were ruled out.

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Conflict of interest statement

Competing Interests: CG & RP are employees of Trophos. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1

Figure 1. Respiration was similar in brain mitochondrial preparations from 24-month-old PARK2−/− and wild type mice examined with standard oxygraphy.

(A) Representative experiment with a crude mitochondrial pellet from striatum. Blue curve = oxygen (O2) concentration expressed as nmol/mL; red curve = rate of oxygen consumption expressed as nmoles O2/mL and min; ADP = addition of 400 µM ADP; OM = addition of 1 µg/mL oligomycin, a complex V inhibitor; KCN = addition of 1 mM KCN (non-respiratory oxygen consumption); (B, C, D) Bars showing state 3 respiration (respiration in the presence of substrates +ADP – non-respiratory oxygen consumption) and state 4 respiration (respiration in the presence of oligomycin – non-respiratory oxygen consumption) on complex I substrates (glutamate+malate); black bars = PARK2−/−, white bars = wild type; data are expressed as nmoles O2/mL and mg proteins and are shown as means ± SD; two animals, one per genotype, were analyzed the same day; the numbers between brackets indicate the number of individual animals of each genotype; (B) results from crude mitochondrial pellets from striatum (n = 6), (C) crude mitochondrial pellets from cortex (n = 7) and (D) purified mitochondria from whole brain (n = 2).

Figure 2

Figure 2. High resolution respirometry reveals a respiration defect in PARK2−/− mice.

(A) Representative experiment with striatal post-nuclear supernatant. Blue curve = oxygen concentration (O2) expressed as nmol/mL; red curve = rate of oxygen consumption expressed as nmoles O2/mL and min; successive additions: ADP = 1 mM ADP in the medium with 10 mM glutamate+5 mM malate; OM = 1 µg/mL oligomycin, cccp = successive additions of 1.25 µM (up to the maximal respiration rate independent from ATP synthase capacity); KCN = 1 mM KCN (non-respiratory oxygen consumption); (B, C, D) Bars showing state 3 respiration, state 4 respiration and respiratory reserve on complex I substrates (glutamate+malate) in post-nuclear supernatants from (B) striatum, (C) midbrain and (D) liver of 24- and 9-month-old PARK2−/− (black bars) and wild type mice (white bars); all data are expressed as nmoles O2/mL and mg proteins and shown as means ± SD; two animals, one per genotype, were analyzed the same day; the data were obtained from 6 to 8 individual animals of each genotype. * p<0.05 using Mann and Whitney test. In liver, state 3 respiration rate and respiratory reserve significantly decreased with age in both wild type mice (p = 0.011 and <0.001 when comparing state 3 respiration and respiratory reserve respectively between 9 and 24 months of age using Mann and Whitney test) and in PARK2−/− mice (p = 0.019 and <0.001 when comparing state 3 respiration and respiratory reserve respectively between 9 and 24 months of age using Mann and Whitney test).

Figure 3

Figure 3. Maximal respiration is reduced in striatal neurons from PARK2−/− mice.

Representative experiments with neurons or astrocytes from striatum or cortex: the traces represent the evolution of the respiration rate (in pmol O2/minute) in Seahorse plates seeded with cells from PARK2−/− (black circles) and wild type (white circles) mice. Sequential additions are: oligomycin (OM) (0.25 µg/ml for neurons and 0.5 µg/ml for astrocytes), fccp (FP) (3 µM for cortical neurons and 1 µM for other cells), and rotenone+antimycine (RA) (50 nM rotenone+150 µg/ml antimycin A). Bars below the traces show the means and SD of basal respiration, maximal respiration and spare respiratory capacity (SRC = maximal – basal respiration) of 3 independent tests, each performed in 6 to 12 independent wells. * p<0.05 using Mann and Whitney test.

Figure 4

Figure 4. The inner mitochondrial membrane potential is normal in midbrain and striatum of PARK2 −/− mice.

Dark circles = PARK2−/−, white circles = wild type; ΔΨm = inner mitochondrial membrane potential expressed as mV after transformation of the fluorescent rhodamine 123 signal using the Nernst equation, as explained in the methods section; A = ΔΨm in the presence of glutamate+malate; B = A+1 mM ADP; C = B+1 µM oligomycin; D = C+1 µM cccp; E = C+2 µM cccp; F = C+4 µM cccp;G = C+8 µM cccp. The data were obtained in parallel to the analysis of respiration; two animals, one per genotype, were analyzed the same day; the data obtained from seven 9-month-old mice and six 24-month-old ones of each genotype, they are expressed as mean and SD.

Figure 5

Figure 5. Increase of the mitochondrial glutathione content in striatum of 12-month-old PARK2 −/− mice.

Total glutathione (GSH+GS-SG) levels were determined in cytosols and isolated mitochondria from ventral midbrain and striatum of 12- and 24-month-old PARK2−/− (black bars) and wild-type mice (white bars), data are expressed as mean and SD of five independent measurements for each age and each genotype. * = p<0.05 using multiple comparison with Holm-Sidak test. Significant differences with age, without influence of the genotype, were observed for midbrain mitochondrial and striatal cytosolic glutathione content. Significant interaction between age and genotype was present for striatal mitochondrial glutathione content, which significantly increased with age only in wild type mice and was at 12 months of age significantly higher in PARK2−/− than in wild type mice.

Figure 6

Figure 6. Presence of oxidative adducts in striatal crude mitochondrial pellets from PARK2 −/− mice.

Western blot analysis of proteins from crude mitochondrial pellets from striatum (A) and midbrain (B) of PARK2 −/− and wild-type mice, at 12 and 24 months of age; loading control was the outer membrane protein Voltage-Dependent Anion Channel (VDAC); (C) densitometric analysis of oxidative adducts was performed in the length of the membrane shown in A and B, that signal was then normalized to VDAC signal and quantification was expressed as means and SD and as % of the mean of wild-type samples on the membrane shown in A and B; * = p<0.05 using Mann and Whitney test.

Figure 7

Figure 7. Increased mitochondrial Mn superoxide dismutase (SOD2) in the striatum of PARK2 −/− mice.

Western blot analysis of proteins from crude mitochondrial pellets from striatum (A) and midbrain (B) of PARK2 −/− and wild-type mice, at 12 and 24 months of age; loading control was the outer membrane protein Voltage-Dependent Anion Channel (VDAC); (C) densitometric analysis of the SOD2 signal shown in A and B was normalized to VDAC signal and quantification was expressed as means and SD and as % of the mean of wild-type samples on the membrane shown in A and B; * = p<0.05 using Mann and Whitney test; † and ‡: samples from 12-month-old PARK2−/− and wild type mice respectively showing that the SOD2 steady-state level does not significantly change with age.

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This work was supported by Institut national de la santé et de la recherche médicale, Association France Parkinson, Fondation de France, Agence Nationale de la Recherche, MEFOPA (funded by the EU 7th Framework Programme, Grant Agreement HEALTH-2009-241791), Fondation ICM, “Investissements d'avenir” ANR-10-IAIHU-06. CG was supported by a fellowship from the Fondation pour la Recherche Médicale. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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