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

<p>(<b>A</b>) 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>B, C, D</b>) 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 = <i>PARK2^−/−</i>, 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).</p

The inner mitochondrial membrane potential is normal in midbrain and striatum of <i>PARK2</i>^−/− mice.

<p>Dark circles = <i>PARK2^−/−</i>, 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.</p

High resolution respirometry reveals a respiration defect in <i>PARK2^−/−</i> mice.

<p>(<b>A</b>) 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>B, C, D</b>) 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 <i>PARK2^−/−</i> (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. * <i>p</i><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 <i>PARK2^−/−</i> 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).</p

Maximal respiration is reduced in striatal neurons from <i>PARK2^−/−</i> mice.

<p>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 <i>PARK2^−/−</i> (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. * <i>p</i><0.05 using Mann and Whitney test.</p

Increased mitochondrial Mn superoxide dismutase (SOD2) in the striatum of <i>PARK2</i>^−/− mice.

<p>Western blot analysis of proteins from crude mitochondrial pellets from striatum (A) and midbrain (B) of <i>PARK2</i>^−/− 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 <i>PARK2^−/−</i> and wild type mice respectively showing that the SOD2 steady-state level does not significantly change with age.</p

Activities of mitochondrial respiratory chain complexes in 12-month-old <i>PARK2^−/−</i> and wild type mice.

<p>Striatum = post-nuclear supernatants from striatum, Midbrain = crude mitochondrial pellet from ventral midbrain, Cortex = crude mitochondrial pellet from occipital cortex; complex I = rotenone sensitive NADH ubiquinone oxidoreductase, complex II = succinate ubiquinone oxidoreductase, combined II+III = succinate cytochrome c oxidoreductase, complex III = antimycin sensitive ubiquinol cytochrome c oxidoreductase, complex IV = cytochrome c oxidase, complex V = F₁F₀ ATPsynthase; complex V assay requires mitochondrial preparation and was therefore not performed on striatal post-nuclear supernatants; data are expressed as nanomoles/minute and mg proteins and as means ± SD; each series of values represents 7 independent samples from each genotype.</p

Proteasome activities were similar in <i>PARK2^−/−</i> and wild type mice.

<p>Chymotrypsin-like activity of the proteasome. Results are expressed as nmoles of substrate cleaved/minute and milligram of protein and as mean±standard deviation of n = 5 measurements.</p

Presence of oxidative adducts in striatal crude mitochondrial pellets from <i>PARK2</i>^−/− mice.

<p>Western blot analysis of proteins from crude mitochondrial pellets from striatum (A) and midbrain (B) of <i>PARK2</i>^−/− 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.</p

Increase of the mitochondrial glutathione content in striatum of 12-month-old <i>PARK2</i>^−/− mice.

<p>Total glutathione (GSH+GS-SG) levels were determined in cytosols and isolated mitochondria from ventral midbrain and striatum of 12- and 24-month-old <i>PARK2^−/−</i> (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 <i>PARK2^−/−</i> than in wild type mice.</p