Parkinson's disease mutations in PINK1 result in decreased Complex I activity and deficient synaptic function

Mutations of the mitochondrial PTEN (phosphatase and tensin homologue)-induced kinase1 (PINK1) are important causes of recessive Parkinson disease (PD). Studies on loss of function and overexpression implicate PINK1 in apoptosis, abnormal mitochondrial morphology, impaired dopamine release and motor deficits. However, the fundamental mechanism underlying these various phenotypes remains to be clarified. Using fruit fly and mouse models we show that PINK1 deficiency or clinical mutations impact on the function of Complex I of the mitochondrial respiratory chain, resulting in mitochondrial depolarization and increased sensitivity to apoptotic stress in mammalian cells and tissues. In Drosophila neurons, PINK1 deficiency affects synaptic function, as the reserve pool of synaptic vesicles is not mobilized during rapid stimulation. The fundamental importance of PINK1 for energy maintenance under increased demand is further corroborated as this deficit can be rescued by adding ATP to the synapse. The clinical relevance of our observations is demonstrated by the fact that human wild type PINK1, but not PINK1 containing clinical mutations, can rescue Complex 1 deficiency. Our work suggests that Complex I deficiency underlies, at least partially, the pathogenesis of this hereditary form of PD. As Complex I dysfunction is also implicated in sporadic PD, a convergence of genetic and environmental causes of PD on a similar mitochondrial molecular mechanism appears to emerge.

Real-time imaging of mitochondrial membrane potential. For evaluation of membrane potential, fibroblast cells were grown in 3cm plastic dishes with glass coverslips (Nunc) and after 424h were loaded with 10nM TMRM (Molecular Probes) in the presence of 2mg/ml cyclosporine H (Sigma) or cyclosporine A, for 30 min at 37°C. Subsequently cells were placed on the stage of an Olympus IX81 inverted microscope equipped with a CellR Imaging system. Sequential images of TMRM fluorescence were acquired every 60s using exposure times of 40ms with a 40x, 1.4 NA Plan Apo objective (Olympus), a 525 ± 20 excitation filter and an emission 570 LP filter.
Flow cytometry. For evaluation of apoptosis, 5x10 5 cells were grown in 12-well plates and after 24h cells were treated with increasing concentrations of H 2 O 2 and Arachidonic acid in HBSS supplemented with 10mM HEPES buffer. After 2h incubation at 37°C cells were harvested and stained with propidium iodide (PI) and Annexin-V-FITC (Invitrogen) according to manufacturer's protocol. Cells were then analyzed by flow cytometry using with a FACSCalibur cytometer (Becton-Dickinson). Viability was measured as the percentage Annexin-V, PI negative cells.
Morais et al.

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Mitochondrial isolation. Mitochondria were isolated from fibroblast cells and from 2 month old mouse liver and brain by standard differential centrifugation and resuspended in Isolation buffer (IB: 0.2M sucrose, 10 mM Tris-MOPS pH 7.4, 0.1 mM EGTA-Tris pH 7.4) as previously described (Frezza et al., 2007).
Fly heads (brain enriched) and thoraxes (muscle enriched) were collected by vigorously shaking approximately 250 flies snap frozen in liquid nitrogen in a double sieve (top sieve 710 µm pore size, bottom sieve 300 µm pore size). Fly bodies are retained on the top sieve, wings and heads on the bottom sieve, and legs fall through. Thoraxes were manually separated from the abdomens, while wings were blown away gently. Thorax and head tissue was then homogenized and mitochondrial homogenates were purified as described by (Schwarze et al., 1998;Walker et al., 2006).
Cytochrome c release ELISA. Cytochrome c release in response to recombinant p7/p15 BID was determined as previously described (Frezza et al., 2006). Briefly, 50µg mitochondria were treated with recombinant p7/p15 BID (32 pMol/mg mitochondria) for the indicated times at 25°C. Cytochrome c release is reported as the percentage of cytochrome c in the supernatant over the total (pellet plus supernatant).
Respiratory Assays. Mitochondrial oxygen consumption was measured by using a Clarke-type oxygen electrode (Hansatech Instruments). Liver mitochondria were incubated in experimental buffer (EB: 125 mM KCl, 10 mM Tris-MOPS, 1 mM KPi, 10 μM EGTA-Tris, pH 7.4, 25°C) supplemented with 5 mM glutamate/2.5 mM malate for analysis of Complex I-driven respiration, 5 mM succinate in the presence of 2 μM rotenone for Complex II-driven respiration, or 3 mM ascorbate plus 150 μM TMPD in the presence of 0.5 μg/ml antimycin A for Complex IV-driven respiration. ADP and FCCP were added at a final concentration of 150µM and 10µM, respectively.
Oxidative phosphorylation complex measurements performed on mitochondrial homogenates from fibroblast cells, mouse brain, fly brain and muscle enriched tissue were analyzed by spectrophotometric assays as previously described (de Paepe et al., 2006). Briefly, measurements of Complex I

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Values were plotted according to the ratio between the specific complex's activity and citrate synthase acitivity. For statistical analysis, all measurements were analyzed using a Student's t test.
Blue Native Gel Electrophoresis. Mitochondria were isolated from 150mg skeletal muscle from Pink1 +/+ and Pink1 -/mouse. Samples were analyzed on a polyacrylamide gel (75µg mitochondrial proteins) and Complexes were separated by BN-PAGE using non-denaturing conditions as described previously (Van Coster et al., 2001). For further Western blot analysis, the gel was transferred onto polyvinylidene difluoride membranes. The blot was de-stained for 1h in distilled water/methanol/acetic acid (40/50/10) and probed with specified antibody.
Mitochondria enriched fractions were prepared as previously mentioned, and 5µg total protein was analyzed on a 4-12% Bis-Tris NuPAGE gel (Invitrogen). The SDS-PAGE was followed by Western blot analysis using the specified primary antibody followed by the horse-radish peroxidase-conjugated secondary antibody. Visualization was performed using the Renaissance chemiluminescence detection system (Perkin-Elmer).
Antibodies. Monoclonal antibodies against the Complex II subunit 70 kDa, the Complex III subunit Core 2, the Complex IV subunit COXIII, the Complex I subunit GRIM19, 20 kDa subunit, NDUFS4, NDUFA9, NDUFS3 were from MitoSciences. The monoclonal antibody Hsp60, a mitochondria matrix protein, was from BD Biosciences and the monoclonal antibody against the Complex I subunit NDUFV1 was from Abcam. For detection of Complex I on BN-PAGE, antibodies against the 20 kDa subunit were used.

Co-immunoprecipitation.
Complex I was co-immunoprecipitated from solubilised mitochondrial proteins as previously described (Keeney et al., 2006). Briefly, 500µg of mitochondrial fraction protein treated with n-dodecyl-β-maltoside, was incubated with Complex I Capture Matrix (MitoSciences). Immunocaptured proteins were eluted with 1% SDS and further analyzed by SDS-PAGE followed by Western blot using the mouse TrueBlot (eBiosciences) as secondary antibody. Bands representing the different complexes are indicated by arrows. Note that in (a) different migration patterns were not observed, and in (b) different Complex I subunits were not detected.
(c)-Mitochondria enriched fractions from Pink1 +/+ and Pink1 -/mouse fibroblasts were analyzed by SDS-PAGE followed by Western blot against the following proteins: GRIM19, a Complex I protein; 70 5 kDa subunit, a Complex II protein; Core 2, a Complex III protein; COX III, a Complex IV protein; and Hsp60, a mitochondrial matrix protein. Hsp60 was used as a control. Note that no significant difference was observed.