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, 105 (32), 11364-9

Loss of PINK1 Causes Mitochondrial Functional Defects and Increased Sensitivity to Oxidative Stress

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Loss of PINK1 Causes Mitochondrial Functional Defects and Increased Sensitivity to Oxidative Stress

Clement A Gautier et al. Proc Natl Acad Sci U S A.

Abstract

Parkinson's disease (PD) is a common neurodegenerative disorder thought to be associated with mitochondrial dysfunction. Loss of function mutations in the putative mitochondrial protein PINK1 (PTEN-induced kinase 1) have been linked to familial forms of PD, but the relation of PINK1 to mammalian mitochondrial function remains unclear. Here, we report that germline deletion of the PINK1 gene in mice significantly impairs mitochondrial functions. Quantitative electron microscopic studies of the striatum in PINK1(-/-) mice at 3-4 and 24 months revealed no gross changes in the ultrastructure or the total number of mitochondria, although the number of larger mitochondria is selectively increased. Functional assays showed impaired mitochondrial respiration in the striatum but not in the cerebral cortex at 3-4 months of age, suggesting specificity of this defect for dopaminergic circuitry. Aconitase activity associated with the Krebs cycle is also reduced in the striatum of PINK1(-/-) mice. Interestingly, mitochondrial respiration activities in the cerebral cortex are decreased in PINK1(-/-) mice at 2 years compared with control mice, indicating that aging can exacerbate mitochondrial dysfunction in these mice. Furthermore, mitochondrial respiration defects can be induced in the cerebral cortex of PINK1(-/-) mice by cellular stress, such as exposure to H(2)O(2) or mild heat shock. Together, our findings demonstrate that mammalian PINK1 is important for mitochondrial function and provides critical protection against both intrinsic and environmental stress, suggesting a pathogenic mechanism by which loss of PINK1 may lead to nigrostriatal degeneration in PD.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Normal ultrastructure but increased numbers of larger mitochondria in the striatum of PINK1−/− mice. (A) Absence of gross ultrastructural defects in mitochondria of the PINK1−/− striatum at 3–4 (upper panels) and 24 (lower panels) months. Arrows indicate the presence of clearly defined and intact cristae. Asterisks show the presence of outer membranes. (B) Distribution of mitochondria in different size bins reveals increased numbers of larger mitochondria in the PINK1−/− striatum at 3–4 and 24 months (*, P < 0.05). (C) Western blot analysis of mitochondrial marker proteins shows normal levels of porin (for outer membrane), AIF (for intermembrane space), Complex V (CxV for inner membrane) and ICDH (for matrix) in the PINK1−/− striatum. Bands shown are representative of a total of 4 samples per genotype.
Fig. 2.
Fig. 2.
Impaired mitochondrial respiration in the PINK1−/− striatum. (A) Representative oxygraphs of striatal mitochondria preparations for complex I, complex II and complex III/IV at 3–4 months of age. After injection of limiting amounts of ADP (indicated by arrows) state 3 was measured. After exhaustion of ADP, O2 consumption slowed down representing state 4 of respiration. (B) State 3 respiratory activity was reduced for both complex I (n = 6) and complex II (n = 9) in PINK1−/− mice. (C) State 4 respiratory activity was reduced for complex I in PINK1−/− mice (n = 6) (*, P < 0.05). (D) Unchanged levels of complex I, complex II and complex III in the striatum at 3–4 months as measured by western blot. (E) Enzymatic activity of aconitase (acon.), malate dehydrogenase (MDH), cytochrome c oxidase (CxIV) and glucose-6-phosphate kinase (G6PK) measured from striatal lysates are shown as normalized to the citrate synthase activity. Aconitase activity was significantly lower in the PINK1−/− striatum (**, P < 0.005, n = 6), whereas other mitochondrial enzyme activities were unaffected. (F) Striatal ATP concentrations are similar in the striatum of PINK1−/− and wild-type mice.
Fig. 3.
Fig. 3.
Age-dependent impairment of mitochondrial respiration in the PINK1−/− cortex. (A) Representative oxygraphs of cortical mitochondria preparations for complex I, complex II and complex III/IV at 3–4 months of age. (B–D) State 3 (B) and state 4 (C) respiratory activities as well as respiratory control ratio (RCR) (D) were normal in the PINK1−/− cortex for all complexes. (E) Representative oxygraphs of cortical mitochondria preparation for complex I, complex II and complex III/IV at 22–24 months of age. (F) State 3 respiratory activity was reduced for all three complexes in the PINK1−/− cortex (*, P < 0.05, n = 4–6). (G) Contrary to state 3, state 4 respiratory activity was increased for complex I in the PINK1−/− cortex. (H) RCR is reduced for complex I and complex II in the PINK1−/− cortex.
Fig. 4.
Fig. 4.
Unchanged levels of oxidative stress markers in PINK1−/− mice. (A) Levels of lipid peroxidation in the mitochondrial fraction in the striatum and cortex measured by the TBARS assay were not significantly (N.S.) different between PINK1−/− and wild-type mice at 2–3 months (n = 6–10 for the cortex, P > 0.05; n = 4 for the striatum, P > 0.05; two-way ANOVA followed by Tukey's test). However, independently of genotype, there was significantly more lipid peroxidation in the striatum compared with the cortex (*, P < 0.025; **, P < 10−3). (B) Oxyblot analysis of striatal lysates at 22–24 months showed similar levels of protein carbonyls in both genotypes. Lanes shown are representative of a total of 6 samples per genotype. (C) 4HNE staining of the entire brain (Upper) and the substantia nigra (Lower) at 22–24 months appeared similar in both genotypes. (D) ROS production as measured by the Amplex red dye assay. Both genotypic groups showed similar ability to produce ROS either under basal conditions or in the presence of the ROS production enhancing toxins, paraquat, 6-OHDA and rotenone. (E) Western blot analysis of antioxidant proteins (Catalase, G6-PDH, SOD1 and 2) and GFAP in the striatum at 22–24 months. Representative results from six PINK1−/− and five wild-type mice are shown. Densitometric analysis shows no significant differences for all proteins tested.
Fig. 5.
Fig. 5.
Increased sensitivity of cortical mitochondria to oxidative stress in PINK1−/− mice. (A) The reduction of state 3 respiration by complex I after 15 min of exposure to 100 μM H2O2 was more dramatic in mitochondria isolated from the PINK1−/− cortex at 3–4 months than from the control (*, P < 0.05, n = 5). Data are expressed as a percentage of the activity of the same sample measured previously in standard conditions. (B) Effect of 15 min of exposure to 500 μM H2O2 on complex II respiratory capacity of cortical mitochondrial preparation at 3–4 months. (*, P < 0.05, n = 9). Data are expressed as a percentage of the activity of the same sample measured previously in standard conditions. (C) Effect of exposure to a mild heat shock (10 min at 43°C before assay) on complex II respiratory capacity of cortical mitochondria preparation at 3–4 months. Data are normalized to WT activity under standard conditions (*, P < 0.05, n = 6). (D) Measure of the transmembrane potential of isolated mitochondria at 3–4 months after 15 min of exposure to 100 μM H2O2 by the JC-1 assay. (E) Mitochondria swelling assay. Data are fitted as the sum of exponentials. τ1 is given as the time constant of the first and main exponential. No difference between genotype was seen under basal conditions or after 15 min of exposure to 200 μM H2O2 (N.S.: not significant, n = 4).

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