The JNK pathway plays a role in the regulation of mitochondrial transport <i>in vivo</i>.

<p>(A) Representative kymographs of mitochondrial transport with overexpression of JNK kinase (Hep^B2) or knockdown of JNK (Bsk RNAi) in response to paraquat. Anterograde transport is toward the right; retrograde transport is toward the left. (B) Paraquat and/or overexpression of Hep^B2 produce a decrease in mitochondrial flux anterogradly. Knockdown of Bsk partially rescues the effect of paraquat. Both overexpression of Hep^B2 and knockdown of Bsk reduce retrograde flux. (C) Anterograde velocity is reduced by paraquat or overexpression of Hep^B2, while retrograde velocity is reduced in both overexpression of Hep^B2 and knockdown of Bsk. (D) Paraquat treatment shows slightly reduction in retrograde duty cycle with a decrease of moving time; Knockdown of Bsk in response to paraquat shows a slightly increase of pause and a decrease of moving time. (E) Retrograde run length is modestly reduced by paraquat treatment. Either overexpression of Hep^B2 of knockdown of Bsk does not show significant difference compared to controls. (F) Paraquat treatment shows an increase of the percentage of stationary mitochondria, while neither overexpression of Hep^B2 nor knockdown of Bsk shows any difference. The number of larvae analyzed is shown on the bars. Error bars indicate mean ± SEM. Significance is determined by one-way ANOVA with Bonferroni’s post-test. *p<0.05, **p < 0.01, and ***p < 0.001.</p

The percentage of moving mitochondria is reduced in response to ROS and rescued by SOD overexpression <i>in vitro</i>.

<p>(A) Representative kymographs of mitochondrial transport under H₂O₂ treatment. Anterograde transport is toward the right; retrograde transport is toward the left. (B) The percentage of moving mitochondria is reduced with H₂O₂ treatment. SOD1 or SOD2 overexpression can rescue the defect. The number of cells analyzed is shown on the bars. Error bars indicate mean ± SEM. Significance is determined by one-way ANOVA with Bonferroni’s post-test. *p<0.05 and ***p < 0.001.</p

The JNK pathway plays a role in the regulation of mitochondrial transport <i>in vivo</i>.

<p>(A) Representative kymographs of mitochondrial transport with overexpression of JNK kinase (Hep^B2) or knockdown of JNK (Bsk RNAi) in response to paraquat. Anterograde transport is toward the right; retrograde transport is toward the left. (B) Paraquat and/or overexpression of Hep^B2 produce a decrease in mitochondrial flux anterogradly. Knockdown of Bsk partially rescues the effect of paraquat. Both overexpression of Hep^B2 and knockdown of Bsk reduce retrograde flux. (C) Anterograde velocity is reduced by paraquat or overexpression of Hep^B2, while retrograde velocity is reduced in both overexpression of Hep^B2 and knockdown of Bsk. (D) Paraquat treatment shows slightly reduction in retrograde duty cycle with a decrease of moving time; Knockdown of Bsk in response to paraquat shows a slightly increase of pause and a decrease of moving time. (E) Retrograde run length is modestly reduced by paraquat treatment. Either overexpression of Hep^B2 of knockdown of Bsk does not show significant difference compared to controls. (F) Paraquat treatment shows an increase of the percentage of stationary mitochondria, while neither overexpression of Hep^B2 nor knockdown of Bsk shows any difference. The number of larvae analyzed is shown on the bars. Error bars indicate mean ± SEM. Significance is determined by one-way ANOVA with Bonferroni’s post-test. *p<0.05, **p < 0.01, and ***p < 0.001.</p

Ca²⁺ homeostasis is required for normal mitochondrial transport.

<p>(A) Representative Ca²⁺ imaging with H₂O₂ or EGTA/BAPTA treatment is measured by the intensity of GCaMP6 indicator. Scale bars indicate 10 μm. (B) Quantitative results from (A). Ca²⁺ levels are increased by H₂O₂ but reduced with EGTA/BAPTA treatment. (C) Representative kymographs of mitochondrial transport with H₂O₂ or EGTA/BAPTA treatment. Anterograde transport is toward the right; retrograde transport is toward the left. (D) EGTA/BAPTA treatment produces a decrease of mitochondrial transport. The reduced percentage of moving mitochondria by H₂O₂ is further reduced by EGTA/BAPTA treatment. The number of cells analyzed is shown on the bars. Significance is determined by one-way ANOVA with Bonferroni’s post-test. **p < 0.01, and ***p < 0.001.</p

Mitochondrial length, membrane potential, and transport are interrelated in response to ROS.

<p>(A) Representative images of mitochondrial length and membrane potential. Mitochondrial lengths are measured using mitoGFP signals and mitochondrial membrane potential is measured using TMRM staining by the intensity ratio of mitochondrial fluorescence to cytosolic fluorescence. Scale bars indicate 10 μm. (B) Quantitative results of mitochondrial length. ROS treatment shows a decrease of mitochondrial length, which is rescued by SOD1 or SOD2 overexpression. (C) Quantitative results of mitochondrial membrane potential. Mitochondrial membrane potential is reduced under oxidative stress conditions. SOD1 or SOD2 overexpression does not rescue these defects. (D) Representative images of mitochondrial length measured using the mitoGFP signal. Scale bars indicate 10 μm. (E) H₂O₂ and/or EGTA/BAPTA reduce mitochondrial length, which is consistent with the results of mitochondrial transport (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178105#pone.0178105.g004" target="_blank">4K, 4L</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178105#pone.0178105.g005" target="_blank">5C and 5D</a>). The number of cells analyzed is shown on the bars. Error bars indicate mean ± SEM. Significance is determined by one-way ANOVA with Bonferroni’s post-test. *p<0.05, **p < 0.01, and ***p < 0.001.</p

ROS changes mitochondrial motility mainly by reducing flux and velocity <i>in vivo</i>.

<p>(A) Representative kymographs of mitochondrial transport under paraquat treatment. Anterograde transport is toward the right; retrograde transport is toward the left. (B) Paraquat treatment produces a decrease in mitochondrial flux, which is rescued by overexpression of SOD1 or SOD2 in both directions. (C) Velocity is reduced with paraquat treatment, and this is rescued by SOD1 or SOD2 overexpression in both directions. (D) Paraquat treatment shows a small reduction in retrograde duty cycle with an increase of pause time and a decrease of moving time; overexpression of SOD1 shows a small increase of pause time. (E) Retrograde run length is modestly reduced with paraquat treatment. (F) Paraquat treatment shows an increase of the percentage of stationary mitochondria, which is rescued by SOD overexpression. The percentage of anterograde moving mitochondria increases with SOD2 overexpression. (G) Mitochondrial density is comparable to control with paraquat treatment or SOD overexpression. The number of larvae analyzed is shown on the bars. Error bars indicate mean ± SEM. Significance is determined by one-way ANOVA with Bonferroni’s post-test. *p<0.05, **p < 0.01, and ***p < 0.001.</p

ROS regulation of axonal mitochondrial transport is mediated by Ca²⁺ and JNK in <i>Drosophila</i>

<div><p>Mitochondria perform critical functions including aerobic ATP production and calcium (Ca²⁺) homeostasis, but are also a major source of reactive oxygen species (ROS) production. To maintain cellular function and survival in neurons, mitochondria are transported along axons, and accumulate in regions with high demand for their functions. Oxidative stress and abnormal mitochondrial axonal transport are associated with neurodegenerative disorders. However, we know little about the connection between these two. Using the <i>Drosophila</i> third instar larval nervous system as the <i>in vivo</i> model, we found that ROS inhibited mitochondrial axonal transport more specifically, primarily due to reduced flux and velocity, but did not affect transport of other organelles. To understand the mechanisms underlying these effects, we examined Ca²⁺ levels and the JNK (c-Jun N-terminal Kinase) pathway, which have been shown to regulate mitochondrial transport and general fast axonal transport, respectively. We found that elevated ROS increased Ca²⁺ levels, and that experimental reduction of Ca²⁺ to physiological levels rescued ROS-induced defects in mitochondrial transport in primary neuron cell cultures. In addition, <i>in vivo</i> activation of the JNK pathway reduced mitochondrial flux and velocities, while JNK knockdown partially rescued ROS-induced defects in the anterograde direction. We conclude that ROS have the capacity to regulate mitochondrial traffic, and that Ca²⁺ and JNK signaling play roles in mediating these effects. In addition to transport defects, ROS produces imbalances in mitochondrial fission-fusion and metabolic state, indicating that mitochondrial transport, fission-fusion steady state, and metabolic state are closely interrelated in the response to ROS.</p></div

Fluorescent images illustrating the angles tracked for MTR-labeled mitochondrial movement in GFP expressing WT and <i>dotA</i><i>⁻</i><i>L. pneumophila-</i> infected cells (A).

<p>“V” represents the vacuole containing the bacterium, “i” indicates the initial position of the mitochondrion, and “f” indicates the final position of the mitochondrion. The relative frequency of the angle of mitochondrial movement towards or away from the vacuole at 1 hour post infection of WT and <i>dotA⁻ L. pneumophila</i>-infected S2 cells are shown in (B, C). The data are represented as the relative frequencies (in 15° bins) as indicated by the length of the lines displayed over 180°. The origin of all radiating relative frequency lines corresponds to the initial position “i,” as shown in (A), for each mitochondrion. All angle measurements considered included only the smaller θ between the three points; thus allowing all angles to be plotted on a semi-circle. Non-moving mitochondria were excluded from the analysis. The distribution of directions of movement was significantly different between the WT and <i>dotA⁻</i> infected cells at 1 hr post-infection (p<0.01; Kolmogorov-Smirnov two-sample test). Scale bar, 20 µm.</p

Frequency distributions of mitochondrial motility parameters detailed for each time point.

<p>The mitochondrial velocity (A), duty cycle (B), and persistence (C) for WT- and <i>dotA^–</i>infected cells from the experiments summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062972#pone-0062972-g004" target="_blank">figure 4</a> are broken out for 1, 2, 3 and 4 hours point post-infection. Most of the data were found not to follow a normal distribution using the 1-sample Kolmogorov-Smirnov test. The only significant differences in mitochondrial motility found between WT- and <i>dotA^–</i>infected cells (indicated by (*)) were (i) the mitochondrial velocity distribution at 1 hr post-infection (P<0.01) and (ii) the duty cycle distribution at both 2 hr and 3 hr post-infection (P<0.025), using the Kolmogorov-Smirnov two-sample test.</p

Distributions of mitochondrial motility parameters in WT- and <i>dotA^–</i>infected S2 cells during the first 4 hours post-infection.

<p>Mean velocity (A), duty cycle (B), and persistence (C) are shown, data are pooled from 24 (wild type) or 25 (dotA-) movies form 4 separate infection experiments. No differences in distribution were detected that would indicate specific effects of infection on mitochondrial motility. Similar comparisons were made separately for each of the four hourly time points and both bacterial strains (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062972#pone-0062972-g005" target="_blank">Figure 5</a>). The majority of the data proved not to follow normal distributions as determined by a one-sample Kolmogorov-Smirnov test. Only the combined active duty cycle distributions (B) were shown to be significantly different, with WT <i>L. pneumophila-</i> infected S2 cells showing a reduced mitochondrial duty cycle relative to <i>dotA⁻ L. pneumophila-</i> infected cells (P<0.001; Kolmogorov-Smirnov two-sample test).</p