PINK1-Mediated Phosphorylation of Parkin Boosts Parkin Activity in <i>Drosophila</i>

<div><p>Two genes linked to early onset Parkinson's disease, <i>PINK1</i> and <i>Parkin</i>, encode a protein kinase and a ubiquitin-ligase, respectively. Both enzymes have been suggested to support mitochondrial quality control. We have reported that Parkin is phosphorylated at Ser65 within the ubiquitin-like domain by PINK1 in mammalian cultured cells. However, it remains unclear whether Parkin phosphorylation is involved in mitochondrial maintenance and activity of dopaminergic neurons <i>in vivo</i>. Here, we examined the effects of Parkin phosphorylation in <i>Drosophila</i>, in which the phosphorylation residue is conserved at Ser94. Morphological changes of mitochondria caused by the ectopic expression of wild-type Parkin in muscle tissue and brain dopaminergic neurons disappeared in the absence of PINK1. In contrast, phosphomimetic Parkin accelerated mitochondrial fragmentation or aggregation and the degradation of mitochondrial proteins regardless of PINK1 activity, suggesting that the phosphorylation of Parkin boosts its ubiquitin-ligase activity. A non-phosphorylated form of Parkin fully rescued the muscular mitochondrial degeneration due to the loss of PINK1 activity, whereas the introduction of the non-phosphorylated Parkin mutant in <i>Parkin</i>-null flies led to the emergence of abnormally fused mitochondria in the muscle tissue. Manipulating the Parkin phosphorylation status affected spontaneous dopamine release in the nerve terminals of dopaminergic neurons, the survivability of dopaminergic neurons and flight activity. Our data reveal that Parkin phosphorylation regulates not only mitochondrial function but also the neuronal activity of dopaminergic neurons <i>in vivo</i>, suggesting that the appropriate regulation of Parkin phosphorylation is important for muscular and dopaminergic functions.</p></div

Effects of phospho-mutant forms of Parkin on various phenotypes.

a<p>Compared with <i>PINK1^-/-; LacZ</i>. ^bCompared with <i>Parkin^-/-; mRFP</i>. ^cCompared with <i>LacZ</i> at 28-day-old. ^dCompared with <i>PINK1^-/-; LacZ</i> at 28-day-old. N.D., not determined.</p

Parkin phosphorylation regulates mitochondrial morphology and the distribution of DA neurons.

<p>Mock (<b>A, A′, E, E′</b>), WT Parkin (<b>B, B′, F, F′</b>), SA Parkin (<b>C, C′, G, G′</b>) or SE Parkin (<b>D, D′, H, H′</b>) were expressed together with mitoGFP in DA neurons of <i>PINK1^+/+</i> (<b>A–D′</b>) or <i>PINK1^-/-</i> (<b>E–H′</b>) flies using the <i>TH</i> driver. Mitochondria and cell bodies of the PPM1/2 cluster DA neurons were visualized using mitoGFP (green in <b>A–H</b> and white in <b>A′–H′</b>) and anti-TH staining (magenta in <b>A–H</b>), respectively. Images (<b>A–H</b>) are higher magnifications of the boxes shown in (<b>A′–H′</b>). Scale bars = 5 µm in (<b>A–H</b>) and 10 µm in (<b>A′–H′</b>). (<b>i</b>) Graph showing the total lengths of mitochondria observed in the cell bodies. # <i>p</i><0.05 <i>vs</i>. mock in <i>PINK1^+/+</i>, § <i>p</i><0.05 <i>vs</i>. WT or SE in <i>PINK1^-/-</i>, ¶ <i>p</i><0.05 <i>vs</i>. all other genotypes in <i>PINK1^-/-</i>, ** <i>p</i><0.01, N.S., not significant. (<b>j</b>) Graph showing the mean areas of aggregated mitochondria greater than 3 µm in each genotype. # <i>p</i><0.05 <i>vs</i>. all other genotypes in <i>PINK1^+/+</i> or <i>PINK1^-/-</i>, § <i>p</i><0.05 <i>vs</i>. mock in <i>PINK1^+/+</i>. ** <i>p</i><0.001, * <i>p</i><0.05 (Student's <i>t</i>-test).</p

Constitutive expression of SE Parkin impairs motor behavior.

<p>(<b>A</b>) LacZ and WT, SA or SE Parkin were expressed under the control of <i>MHC-GAL4</i> in the <i>w-</i> background. WT and SA Parkin improved the motor activity in older flies, whereas SE Parkin compromised motor activity throughout the trials. LacZ served as a control. The values are presented as the mean ± SE from 20 trials. Male flies were used for the assay. **<i>P</i><0.01 <i>vs</i>. <i>LacZ</i>; #<i>P</i><0.05. Flies expressing SE Parkin had decreased climbing ability compared with flies expressing WT or SA Parkin throughout the trial (<i>P</i><0.01). (<b>B</b>) Muscle-specific expression of Parkin SE under control of <i>MHC-GAL4</i> failed to rescue the motor defect of the <i>PINK1^-/-</i> flies. The loss of climbing ability in the <i>PINK1^-/-</i> flies was rescued by expression of WT and SA Parkin. The values represent the mean ± SE from 20 trials. Male flies were used for the assay. **<i>P</i><0.01 <i>vs</i>. <i>LacZ</i>. Flies expressing SE Parkin had decreased climbing ability compared with flies expressing WT or SA Parkin throughout the trial (<i>P</i><0.01).</p

SE Parkin, but not WT or SA, diminishes complex I integrity.

<p>(<b>A</b>) Parkin (WT, SA or SE) or β-galactosidase (LacZ) was expressed in the thorax muscle using the <i>MHC</i> driver. The indicated mitochondrial proteins from the thoraxes of 14-day-old adult flies were analyzed by western blot. Actin was used as a loading control. Note that SE Parkin expression was reduced in this setting (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004391#s3" target="_blank">Discussion</a>). (<b>B, C</b>) The band intensities of the indicated mitochondrial proteins were normalized to each Actin signal. The values (arbitrary units) represent the mean ± SE from five independent samples as in (<b>A</b>). ** <i>p</i><0.01, * <i>p</i><0.05 <i>vs</i>. <i>LacZ</i>. (<b>D–F</b>) <i>LacZ</i> and <i>Parkin</i> were expressed in <i>PINK1^-/-</i> flies using the <i>MHC</i> driver as in (<b>A–C</b>). <i>LacZ</i> expressed in the <i>w-</i> background was used as a control (control). (<b>E</b>) ** <i>p</i><0.01, * <i>p</i><0.05 <i>vs. control</i> or <i>PINK1^-/-;LacZ</i>. (<b>F</b>) * <i>p</i><0.05 <i>vs. control</i>. (<b>G, H</b>) Effects of increasing amounts of Parkin WT, SA and SE on mitochondrial proteins and Drp1. Parkin was induced in adult flies crossed with the ubiquitous <i>daughterless</i>–Gene-Switch driver (<i>Da-GS</i>) by raising flies with media containing the indicated amounts of RU486 (0, 0.25, 0.5 and 1.0 µg/ml) for 3 days. <i>UAS-LacZ</i> crossed with <i>Da-GS</i> (<i>Da-GS>LacZ</i>) and <i>Parkin WT</i> crossed with <i>MHC-GAL4</i> (<i>MHC>Parkin WT</i>) serve as a negative control and a positive control, respectively. (<b>H</b>) Mfn levels were quantified and normalized to each Actin level in flies treated with (+RU486) or without (- RU486) 0.25 µg/ml RU486 as in (<b>G</b>). * <i>p</i><0.05 <i>vs. WT</i>, +RU486.</p

Parkin phosphorylation modulates dopaminergic function and survivability of DA neurons.

<p>(<b>A</b>) Confocal images of DA neurons (magenta) from the brains of 5-day-old flies expressing VMAT-pHluorin (green) together with the control LacZ and WT, SA or SE Parkin (Left). The <i>TH</i> driver was used for transgene expression. VMAT-pHluorin signals are shown as white signals (Right). Scale bar = 100 µm. (<b>B</b>) The expression of SA or SE Parkin causes early impairment of vesicular dopamine release in the nerve terminals of DA neurons. The fluorescence recovery rate of DA neuron terminals in cultured neural tissue post-photobleaching was used to estimate the spontaneous DA release rate. Data are shown as the mean ± SE. * <i>p</i><0.05 <i>vs.</i> LacZ and WT, # <i>p</i><0.01 <i>vs</i>. WT. n = 15–20. (<b>C</b>) The flying activity of 15- and 22-day-old males expressing LacZ (control) and those expressing WT, SA and SE Parkin using the <i>TH</i> driver. Data are shown as the mean ± SE. ** <i>p</i><0.01 <i>vs</i>. control and WT. n = 10. (<b>D</b>) Quantification of the number of TH⁺ DA neurons in the PPM1/2 clusters in 5- and 25-day-old males expressing LacZ (control) and those expressing WT, SA and SE Parkin using the <i>TH</i> driver. Data are shown as the mean ± SE. ** <i>p</i><0.01 <i>vs</i>. control of the same age, # <i>p</i><0.01, N.S., not significant. n = 17–20 for control, WT and SA at 45 days. n = 5–8 for flies at 5 days and for SE flies at 45 days. (<b>E</b>) The same assay shown in (<b>D</b>) was performed in the <i>PINK1</i>-deficient flies. * <i>p</i><0.05, ** <i>p</i><0.01 <i>vs</i>. control of the same age, # <i>p</i><0.01 <i>vs</i>. control and WT at 45 days, § <i>p</i><0.01 <i>vs</i>. SA at 45 days. n = 17–20 at 45 days. n = 6–9 at 5 days. (<b>F</b>) Survival curves of flies post-eclosion. Expression of WT, SA and SE Parkin in muscle tissues using the <i>MHC</i> driver rescued the shortened lifespan of <i>PINK1</i>-deficient flies (<i>p</i><1⁻²⁰ by log-rank test; n = 93–170 male flies); there were no differences in the rescue effect among WT, SA and SE. (<b>G</b>) SA Parkin (<i>P</i><1⁻¹¹ by log-rank test; n = 146 male flies) or SE Parkin (<i>P</i><1⁻²⁰; n = 223) expression in DA neurons using the <i>TH</i> driver shortened the lifespan compared with the control LacZ (n = 320) and WT Parkin (n = 154).</p

Phosphorylation of the Parkin Ubl domain regulates mitochondrial morphology.

<p>(<b>A</b>) Parkin is phosphorylated by PINK1 in insect cells. S2 cells transfected with the indicated plasmids with or without <i>Drosophila</i> PINK1 were treated with or without 30 µM carbonyl cyanide <i>m</i>-chlorophenylhydrazone (CCCP) for 1 h. The cell lysate was subsequently separated on a Phos-tag gel, followed by western blotting with anti-<i>Drosophila</i> Parkin. (<b>B</b>) The phosphorylation status of Parkin affects the mitochondrial length in muscle tissue. Fluorescent and TEM images of the indirect flight muscle in the indicated genotypes of 14-day-old adult flies are shown. To visualize the mitochondria, the mitoGFP (green) transgene was co-expressed, and the muscle tissue was counterstained with phalloidin (magenta). Mitochondria in the TEM images are outlined with broken lines to highlight their morphology. Scale bars = 10 µm in the fluorescent images and 2 µm in the TEM images. (<b>C</b>) Mitochondrial morphology of 14-day-old <i>PINK1</i> mutant flies expressing mock, WT Parkin and phospho-mutants. The inset shows a high-magnification TEM image of <i>PINK1^-/-; Parkin SE</i> with intact mitochondrial matrices. Scale bars = 10 µm in the fluorescent images and 2 µm in the TEM images. (<b>D</b>) The length of the long axis of the muscle mitochondria was calculated. The data represent the mean ± SE from three flies (<i>n</i> = 25 in each). ** <i>p</i><0.01 <i>vs</i>. all other genotypes, # <i>p</i><0.01 <i>vs</i>. mock with <i>PINK1^+/+</i>, § <i>p</i><0.01 <i>vs</i>. <i>PINK1^RNAi; WT</i> or <i>SA Parkin</i>, * <i>p</i><0.05, N.S., not significant. (<b>E</b>) Mitochondrial morphology of 14-day-old <i>Parkin</i> mutant flies expressing mock, WT or SA Parkin. Scale bars = 10 µm in all fluorescent images and 2 µm for <i>Parkin^-/-</i> and <i>WT Parkin; Parkin^-/-</i> and 5 µm for <i>SA Parkin; Parkin^-/-</i> in the TEM images. Arrowheads indicate large mitochondrial aggregates. (<b>F</b>) The length of the long axis of the muscle mitochondria was calculated. The data represent the mean ± SE from three flies (<i>n</i> = 25 in each). * <i>p</i><0.05. The genotypes are as follows: (<b>B</b>) <i>UAS-mitoGFP/+</i>; <i>MHC-GAL4/+</i> (control), <i>UAS-mitoGFP/UAS-Parkin</i>; <i>MHC-GAL4</i> (<i>WT, SA</i> and <i>SE Parkin</i>). (<b>C</b>) <i>PINK1^B9/Y; UAS-LacZ</i>; <i>MHC-GAL4</i> (<i>PINK1^-/-</i>), <i>PINK1^B9/Y; UAS-Parkin</i>; <i>MHC-GAL4</i> (<i>PINK1^-/-; WT, SA</i> or <i>SE Parkin</i>). <i>UAS-mitoGFP; MHC-GAL4, UAS-PINK1 RNAi</i> crosses were used rather than <i>PINK1^B9</i> crosses for fluorescent images. (<b>E</b>) <i>UAS-mitoGFP/UAS-LacZ</i>; <i>Da-GAL4, Parkin^Δ21/Parkin¹</i> (<i>Parkin^-/-</i>), <i>UAS-mitoGFP/UAS-Parkin</i>; <i>Da-GAL4, Parkin^Δ21/Parkin¹</i> (<i>Parkin^-/-; WT</i> or <i>SA Parkin</i>).</p

Temporal order judgment in the arms-crossed condition.

<p>(A-C) The red symbols indicate the probability of a right-hand first judgment in the arms-crossed condition plotted against the SOA for the young (A), non-PD elderly (B), and PD elderly participants (C). The black and red curves indicate the results of the model fitting in the uncrossed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118331#pone.0118331.e001" target="_blank">Equation 1</a>) and crossed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118331#pone.0118331.e002" target="_blank">Equation 2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118331#pone.0118331.e004" target="_blank">4</a>) conditions. Each symbol represents 144 (A, 8×18), 184 (B, 8×23) and 128 (C, 8×16) trials. The other conventions are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118331#pone.0118331.g001" target="_blank">Fig. 1A</a>. (D-F) The difference between the order-judgment probability in the crossed and uncrossed condition (ordinate) are plotted against the stimulation onset asynchrony (SOA, abscissa). The difference shown in (D), (E), and (F) was calculated using data from the young (A), non-PD elderly (B), and PD elderly participants, respectively. The upward and downward Gaussian curves (blue) correspond to the flip functions, <i>f</i>_l and <i>f</i>_r, of the judgment probabilities as defined in Equations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118331#pone.0118331.e003" target="_blank">3</a>) and (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118331#pone.0118331.e004" target="_blank">4</a>). The net peak flip amplitudes (<i>Ã</i>_<i>l</i> and <i>Ã</i>_<i>r</i> in Equations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118331#pone.0118331.e005" target="_blank">5</a>) and (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118331#pone.0118331.e006" target="_blank">6</a>)) and the probability of generic error (<i>c</i> in Equations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118331#pone.0118331.e003" target="_blank">3</a>) and (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118331#pone.0118331.e004" target="_blank">4</a>)) are indicated in (F). (G-L) Group comparisons: the net left-to-right flip probabilities (<i>Ã</i>_<i>l</i>) (G), those of the reverse (<i>Ã</i>_<i>r</i>) (H), the constant error rate (<i>c</i>) (I), the time window of the flip (<i>σ</i>_<i>f</i>) (J), and error response rates in the catch (K) and the single stimulus trials (L). The box-plot conventions are as Figs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118331#pone.0118331.g001" target="_blank">1D-F</a>. The brackets with asterisks show that the median was significantly different (p < 0.01, Wilcoxon rank sum test after the Bonferroni correction for multiple comparisons). (M) The time window of the flip (<i>σ</i>_<i>f</i>, ordinate) plotted against the constant error rate (<i>c</i>, abscissa). Data from the young (circles), non-PD elderly (squares), and PD elderly participants (triangles) are shown with different symbols. Note a significant correlation between the two parameters (Spearman’s <i>ρ</i> = 0.36, <i>p</i> = 0.0055).</p

Comparisons of reaction time.

<p>The mean reaction time (ordinate) is plotted against the SOA (abscissa) for the young (A), non-PD (B), and PD participants (C). The colors differentiate between the arms-crossed (red) and -uncrossed (black) conditions.</p

Demographics of the participants.

<p>An asterisk (*) indicates a significant difference between the non-PD and PD participants (p < 0.001, Wilcoxon rank sum test). Each cell shows the mean ± s.e.m, where applicable. On/off: on-medication/off-medication. NA: not applicable.</p><p>Demographics of the participants.</p