Phosphorylation of MCAD selectively rescues PINK1 deficiencies in behavior and metabolism

Abstract

PTEN-induced putative kinase 1 (PINK1) is a mitochondria-targeted kinase whose mutations are a cause of Parkinson's disease. We set out to better understand PINK1's effects on mitochondrial proteins in vivo. Using an unbiased phosphoproteomic screen in Drosophila, we found that PINK1 mediates the phosphorylation of MCAD, a mitochondrial matrix protein critical to fatty acid metabolism. By mimicking phosphorylation of this protein in a PINK1 null background, we restored PINK1 null's climbing, flight, thorax, and wing deficiencies. Owing to MCAD's role in fatty acid metabolism, we examined the metabolic profile of PINK1 null flies, where we uncovered significant disruptions in both acylcarnitines and amino acids. Some of these disruptions were rescued by phosphorylation of MCAD, consistent with MCAD's rescue of PINK1 null's organismal phenotypes. Our work validates and extends the current knowledge of PINK1, identifies a novel function of MCAD, and illuminates the need for and effectiveness of metabolic profiling in models of neurodegenerative disease.

FIGURE 1:

Phosphorylation of MCAD S347 is dependent on PINK1 in Drosophila. (A) Phosphopeptide analysis showing relative levels of phosphorylated (left) and unphosphorylated (right) MCAD peptide in mitochondrial fractions from 5-d-old control and PINK1 null male flies. (B) Sequence alignment of human, mouse, rat, and fly MCAD. S347/T351 is marked in red. (C) Phos-tag and regular PAGE gels showing in vitro kinase assay for MCAD and TcPINK1. Phos-tag gel is probed with anti-MCAD to detect both unphosphorylated and the slower-migrating phosphorylated MCAD. Regular PAGE gels are probed with anti-MCAD and anti-MBP to detect MCAD and TcPINK1, respectively, in the in vitro kinase assays. CIP is calf intestinal alkaline phosphatase. The same results were repeated three times (D) Schematic depiction of Drosophila gene CG12262 (MCAD) detailing sequences used to generate wild-type, phosphoresistant and phosphomimetic S347. Green boxes are 5′ and 3′ UTRs; blue boxes are exons. (E) Representative Western blot of mitochondrial and cytosolic fractions from Actin5C-GAL4 alone (control) and Actin5C-GAL4 driving MCAD (A), MCAD (WT), and MCAD (D) transgenic constructs. Samples were probed with anti-tubulin as a cytosolic loading control, anti-OPA1 as a mitochondrial loading control, anti-MCAD to identify endogenous and transgenic localization, and anti-V5 to verify transgenic localization. Note that endogenous MCAD (red asterisk) migrates faster than V5-tagged MCAD in the anti-MCAD blot. The slightly shifted V5-tagged transgenes are verified in the separate anti-V5 blot. n = 25 flies lysed per sample for each experiment; three biological replicates per genotype.

FIGURE 2:

Phosphomimetic MCAD S347 rescues PINK1 null phenotypes. (A–F) All assays were conducted in 3- to 4-d-old male flies. n = 50–55 flies. For all panels, comparison is to PINK1^RV unless otherwise indicated. All lines showing significance represent two-group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for all figures. (A) Time flies take to climb upward 8 cm. One-way ANOVA, followed by Tukey’s multiple comparisons. Middle line is the median, outer edges of the box are 25th and 75th percentiles, and whiskers are the minimum and maximum values. (B) Percentage of flies that cannot fly. (C) Percentage of flies with indented thoraxes. (D) Percentage of flies with drooped or held-up wings. (B–D) Chi-square test, followed by Fisher’s exact tests. (E) Representative images of a healthy PINK1^RV (control) thorax (left), and the indented thorax of a PINK1^B9(null) fly (right). Red arrows indicate indentation. (F) Representative images of healthy PINK1^RV wings (left), as well as drooped (middle) and held-up (right) wings in PINK1^B9flies. Red dotted lines emphasize the varying angles of the wings. (G) ATP levels divided by total protein levels, n = 4–5 samples, four flies per sample. One-way ANOVA, followed by Tukey’s multiple comparisons; n.s., not significant.

FIGURE 3:

Acylcarnitine analysis in PINK1 null and MCAD mutant flies. Acylcarnitine analysis in lysates from 6- to 7-d-old male flies. “Starved” condition means flies were switched to vials containing only water for the final 24 h. Student’s t tests, n = 3–10 samples, five flies per sample, two technical replicates averaged per sample. Abundance of (A) C6, (B) C8, and (C) C10:1 acylcarnitines in MCAD deficient flies and PINK1 null flies.

FIGURE 4:

MCAD S347D selectively rescues PINK1 null’s acylcarnitine and amino acid deficiencies. Comparing selected acylcarnitines and amino acids between PINK1^RV and PINK1^B9, and PINK1^B9; Actin5C-GAL4>MCAD (A) and PINK1^B9; Actin5C-GAL4>MCAD (D). Student’s t tests, n = 3–8 samples, five flies per sample, two technical replicates averaged per sample.