Journal: The Journal of Cell Biology

Article Title: Cardiolipin promotes electron transport between ubiquinone and complex I to rescue PINK1 deficiency

doi: 10.1083/jcb.201511044

Figure Lengend Snippet: Pharmacological inhibition of FASN rescues PINK1-deficient flight defects and ATP levels. (A) Simplified schematic representation of the enzymatic reaction catalyzed by FASN requiring one Acetyl-CoA and seven Malonyl-CoAs to produce one palmitate in a seven-step enzymatic process. Compounds Cerulenin, Irgasan, and Orlistat block FASN at the second, sixth, and seventh steps, respectively. (B–G) Suppression of pink1 B9 phenotypes by supplementing the fly medium with Cerulenin (green bars; B and C), Irgasan (blue bars; D and E), or Orlistat (pink bars; F and G) compared with control fly medium (black bars) for flying ability ( n = 50 flies; B, D, and F) and ATP levels ( n = 10 assays; 100 µM for Cerulenin, 3 µM for Irgasan, and 100 µM for Orlistat; C, E, and G). 1-d-old flies were selected and placed on the medium for 3 d before measuring ATP levels on 4-d-old flies. Student’s t test: *, P < 0.05. Data represent percentages (B, D, and F) or means (C, E, G, and H) ± SEM.

Article Snippet: 1-d-old adult flies were placed on molasses medium supplemented with 100 µM Cerulenin (Enzo Life Sciences), 3 µM Irgasan (Sigma-Aldrich), or 100 µM Orlistat (Sigma-Aldrich), unless otherwise indicated in (B, D, and F; 2.5% final ethanol concentration), or flies were supplemented with 2.5% ethanol for control medium.

Techniques: Inhibition, Blocking Assay

Journal: The Journal of Cell Biology

Article Title: Cardiolipin promotes electron transport between ubiquinone and complex I to rescue PINK1 deficiency

doi: 10.1083/jcb.201511044

Figure Lengend Snippet: The effect of FASN inhibition is evolutionarily conserved. (A–C) Images (A) and quantification (B) of mitochondrial morphology ( n = 20 muscle sections) in muscles of third instar larvae and electron micrographs of adult thorax muscle mitochondria (C; n = 5 flies) after Cerulenin incubation. Arrowheads in A denote mitochondrial aggregation (blebs). Boxes in C denote magnified areas. Bars: (A) 5 µm; (C) 0.5 µm. (D–F) ATP levels of Pink1 −/− MEFs ( n = 5 assays; D), patient fibroblasts ( n = 5 assays of two independent control or patient lines; E), and dopaminergic neurons derived from iPSCs from patients ( n = 3 assays of two independent control or patient lines; F) incubated with 10 µM Cerulenin (green bars) or with control medium (black bars). Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data represent percentages (D–F) or means (B) ± SEM.

Article Snippet: 1-d-old adult flies were placed on molasses medium supplemented with 100 µM Cerulenin (Enzo Life Sciences), 3 µM Irgasan (Sigma-Aldrich), or 100 µM Orlistat (Sigma-Aldrich), unless otherwise indicated in (B, D, and F; 2.5% final ethanol concentration), or flies were supplemented with 2.5% ethanol for control medium.

Techniques: Inhibition, Incubation, Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Cardiolipin promotes electron transport between ubiquinone and complex I to rescue PINK1 deficiency

doi: 10.1083/jcb.201511044

Figure Lengend Snippet: Loss of FASN decreases palmitate levels in Pink1 deficiency. (A and B) Free fatty acid levels are reduced in pink1 B9 mutant flies and are further reduced by heterozygosity of dFasn2 in pink1 B9 mutant flies (pink; A) or by Cerulenin (green) in pink1 B9 mutants (B) compared with pink1 RV control flies (black; n = 5 assays). (C and D) Free fatty acid levels (C) and palmitate levels (D) in Pink1 −/− MEFs incubated with Cerulenin (green) are reduced compared with those not incubated with Cerulenin (black) and to Pink1 +/+ control MEFs. n = 5 assays. (E–J) Quantification (E, G, and I) and images (F, H, and J) of protein levels of FASN normalized to actin in Pink1 −/− MEFs (E and F), patient-derived fibroblasts (G and H), and H 2 O 2 -treated wild-type MEFs (I and J). n = 3 assays. Bonferri’s analysis of variance (A–D) and Student’s t test (E, G, and I): *, P < 0.05; **, P < 0.01; ns, not significant. Data represent percentages (A–D) or normalized means (E, G, and I) ± SEM.

Article Snippet: 1-d-old adult flies were placed on molasses medium supplemented with 100 µM Cerulenin (Enzo Life Sciences), 3 µM Irgasan (Sigma-Aldrich), or 100 µM Orlistat (Sigma-Aldrich), unless otherwise indicated in (B, D, and F; 2.5% final ethanol concentration), or flies were supplemented with 2.5% ethanol for control medium.

Techniques: Mutagenesis, Incubation, Derivative Assay

Journal: The Journal of Cell Biology

Article Title: Cardiolipin promotes electron transport between ubiquinone and complex I to rescue PINK1 deficiency

doi: 10.1083/jcb.201511044

Figure Lengend Snippet: Loss of Pink1 results in decreased CL levels. (A) Palmitate produced by FASN promotes the formation of saturated PG that is a weak substrate for CLS, thus resulting in lower CL levels. Lower levels of palmitate upon inhibition of FASN shift the balance to increased levels of unsaturated PG, which serve as a strong substrate for CLS, thus producing more CL. (B–D) Shotgun lipid analysis of mitochondria isolated from Pink1 −/− and Pink1 +/+ MEFs ( n = 2 independent experiments) that were incubated with Cerulenin (green bars) compared with those incubated with control medium (black bars) for PG separated by side chain unsaturation levels (B), CL alone (C), and CL separated by side chain unsaturation level (D). Student’s t test: *, P < 0.05. Data represent means ± SEM.

Article Snippet: 1-d-old adult flies were placed on molasses medium supplemented with 100 µM Cerulenin (Enzo Life Sciences), 3 µM Irgasan (Sigma-Aldrich), or 100 µM Orlistat (Sigma-Aldrich), unless otherwise indicated in (B, D, and F; 2.5% final ethanol concentration), or flies were supplemented with 2.5% ethanol for control medium.

Techniques: Produced, Inhibition, Isolation, Incubation

Journal: The Journal of Cell Biology

Article Title: Cardiolipin promotes electron transport between ubiquinone and complex I to rescue PINK1 deficiency

doi: 10.1083/jcb.201511044

Figure Lengend Snippet: Loss of FASN improves complex I activity upon Pink1 deficiency. (A and B) Complex I activity normalized to citrate synthase (CS) in pink1 B9 mutant flies that were rescued by heterozygous dFasn2 1 in pink1 B9 mutant flies (pink; A) or by inhibition of FASN by Cerulenin (green; B) in pink1 B9 mutant flies compared with pink1 RV control flies (black). n = 8 assays. (C) Complex I activity normalized to citrate synthase in Pink1 −/− MEFs incubated with Cerulenin (green) compared with those not incubated with Cerulenin (KD; black) and to Pink1 +/+ control MEFs. n = 5 assays. (D) Complex I activity normalized to citrate synthase in cells where NdufA10 was knocked down and where wild-type NdufA10 (WT) or phosphorylation-deficient NdufA10 (S250A) are reexpressed and then incubated with Cerulenin (green) or control medium (black). n = 4 assays. Student’s t test: *, P < 0.05; ns, not significant. Data represent percentages ± SEM.

Article Snippet: 1-d-old adult flies were placed on molasses medium supplemented with 100 µM Cerulenin (Enzo Life Sciences), 3 µM Irgasan (Sigma-Aldrich), or 100 µM Orlistat (Sigma-Aldrich), unless otherwise indicated in (B, D, and F; 2.5% final ethanol concentration), or flies were supplemented with 2.5% ethanol for control medium.

Techniques: Activity Assay, Mutagenesis, Inhibition, Incubation