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. 2011 Jan;41(1):189-200.
doi: 10.1016/j.nbd.2010.09.006. Epub 2010 Sep 17.

Integrating multiple aspects of mitochondrial dynamics in neurons: age-related differences and dynamic changes in a chronic rotenone model

Beth Arnold  1 , Steven J Cassady, Victor S VanLaar, Sarah B Berman
Affiliations

Integrating multiple aspects of mitochondrial dynamics in neurons: age-related differences and dynamic changes in a chronic rotenone model

Beth Arnold et al. Neurobiol Dis. 2011 Jan.

Abstract

Changes in dynamic properties of mitochondria are increasingly implicated in neurodegenerative diseases, particularly Parkinson's disease (PD). Static changes in mitochondrial morphology, often under acutely toxic conditions, are commonly utilized as indicators of changes in mitochondrial fission and fusion. However, in neurons, mitochondrial fission and fusion occur in a dynamic system of axonal/dendritic transport, biogenesis and degradation, and thus, likely interact and change over time. We sought to explore this using a chronic neuronal model (nonlethal low-concentration rotenone over several weeks), examining distal neurites, which may give insight into the earliest changes occurring in PD. Using this model, in live primary neurons, we directly quantified mitochondrial fission, fusion, and transport over time and integrated multiple aspects of mitochondrial dynamics, including morphology and growth/mitophagy. We found that rates of mitochondrial fission and fusion change as neurons age. In addition, we found that chronic rotenone exposure initially increased the ratio of fusion to fission, but later, this was reversed. Surprisingly, despite changes in rates of fission and fusion, mitochondrial morphology was minimally affected, demonstrating that morphology can be an inaccurate indicator of fission/fusion changes. In addition, we found evidence of subcellular compartmentalization of compensatory changes, as mitochondrial density increased in distal neurites first, which may be important in PD, where pathology may begin distally. We propose that rotenone-induced early changes such as in mitochondrial fusion are compensatory, accompanied later by detrimental fission. As evidence, in a dopaminergic neuronal model, in which chronic rotenone caused loss of neurites before cell death (like PD pathology), inhibiting fission protected against the neurite loss. This suggests that aberrant mitochondrial dynamics may contribute to the earliest neuropathologic mechanisms in PD. These data also emphasize that mitochondrial fission and fusion do not occur in isolation, and highlight the importance of analysis and integration of multiple mitochondrial dynamic functions in neurons.

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Figures

Figure 1
Figure 1
Chronic rotenone alters mitochondrial fission and fusion. Primary rat cortical neurons were transfected with mtDsRed2 and PAmtGFP, then imaged under normal culturing conditions at times indicated (B) or treated beginning at DIV7 for 1or 2 weeks with media containing rotenone at listed concentrations or DMSO vehicle control (C-F). Live images were recorded for 15 min as described in Methods, and fission and fusion events were quantified blindly as described (n = at least 7 independent experiments, with 3-6 neuronal processes imaged per condition in each experiment. A, Sequential images showing example of mitochondrial fusion (2nd and 3rd panel: mitochondrion with mtDsRed2 and photoactivated PAmtGFP (yellow) fuses with neighboring mitochondrion (red) with resulting diffusion of GFP into newly fused side) and fission (4th panel: yellow mitochondrion now divides into two) (Also see Supplemental Movie 1). Gray hashed lines outline neuronal process. Scale bar is 2 μm. B, Probability of fission and fusion events occurring in photoactivated mitochondria over 15 min at DIV7 and DIV28 in culture (*Significantly different from DIV28 neurons, p=0.0173, two-tailed binomial test of differences between proportions). C, Probability of fusion events under conditions as noted. *Control vs 1 nM p = 0.0023 two tailed; **0.1 nM vs 1 nM p < 0.002. D, Probability of fission events under conditions noted. *p = 0.011 (binomial test of differences between proportions with Bonferroni correction). E, Change in the ratio of fission events to fusion events after one or two weeks exposure to rotenone (0.1 and 1 nM) compared to DMSO control. F, Percentage of events in which an individual mitochondrion underwent a fusion event followed by at least one fission event in a 15 min observation, after one or two weeks of rotenone or vehicle control (+/− SE). *95% confidence interval of proportions different from control.
Figure 2
Figure 2
Mitochondrial transport after chronic rotenone. Cortical neurons transfected and treated with chronic rotenone as in Figure 1 were imaged every 10 s for 15 min. A-C, Example of manual tracking using Image J with Manual Tracking plug-in as described. A, Fluorescent mitochondria in neurite. B, Mitochondria are identified, and as shown in (C), are tracked throughout the image series. D, Overall average individual mitochondrial transport velocity. E, Ratio of retrograde-moving mitochondria to anterograde-moving mitochondria in each condition. *Total proportion of retrograde mitochondria in motile population (59/110 in control vs. 58/84 in 0.1 nM rotenone different than control by two-tailed test of proportions as described) and the total proportion of non-moving mitochondria in each condition (no differences). F-G, Relationship of mitochondrial length and transport velocity. Distribution of mitochondrial velocity by individual mitochondrial length under conditions as listed.
Figure 3
Figure 3
Effect of chronic rotenone on mitochondrial morphology. Cortical neurons were transfected and treated with rotenone or DMSO vehicle control as described in previous figures. Randomly identified neuronal processes were imaged as described above. Image J was utilized to identify fluorescent mitochondria, and length was measured. A, One week rotenone or control exposure. B, Two week exposure. *Significantly different than control (p = 0.003). C-D, Using the manual tracking procedure as in Figure 2, mitochondria were separated into those traveling anterograde or retrograde (non-moving mitochondria not included in this analysis), and average length calculated after 1 (C) or 2 (D) week exposure to rotenone or vehicle control. There are no significant differences between groups.
Figure 4
Figure 4
Mitochondrial density after chronic rotenone exposure. Cortical neurons were treated as described previously, and images taken as described in Methods. A, Example of 3D Metamorph reconstruction of fluorescent mitochondria in neuronal cell body for volume measurements. B, Example of soluble GFP-expressing neurites with mtDsRed2 in distal process. C-D, Mitochondrial density in cell body, proximal neurites, or distal neurites after one or two weeks of rotenone exposure or vehicle control. *Rotenone treatment conditions significantly different than vehicle control. In addition, there are age-related changes, unmarked in graph: control-treated neuronal cell bodies have greater mitochondrial density at the two week time point (DIV21) than at the one week time point (DIV14; p=0.005), but are not statistically significantly different in the other regions at those time points.
Figure 5
Figure 5
Measures of autophagy and mitophagy in neurons after chronic rotenone. Cortical neurons were transfected with mtDsRed2, treated at DIV7 with rotenone (1 nM) or vehicle control for one or two weeks as described in Methods, then fixed. Immunocytochemistry was performed to stain the autophagic vesicle marker LC3. A, Representative neuronal cell body image (compressed z-stack). B, Percentage of neurons in listed conditions containing greater than 4 autophagic puncta (no significant differences; 5 individual experiments, 64-99 neurons/condition). C, Colocalization of mitochondria with LC3-containing puncta (Pearson’s colocalization coefficients of red and green fluorescence, calculated from each individual z-slice; 3 individual experiments with 5 neurons per condition per experiment; no significant differences).
Figure 6
Figure 6
Effect of chronic rotenone on differentiated dopaminergic PC6-3 cells. Cells were differentiated for 7d with 40 ng/ml NGF, and then treated with rotenone or DMSO vehicle for an additional 7,14, or 21 days. A, Effect of rotenone concentration on cell viability over time. **1 week, #2 week, or *3 week significantly different from control (ANOVA with Bonferroni corrections to p < 0.005; n= 4 individual experiments in triplicate). B, Percentage of long processes in cells after chronic rotenone over time (assessed as those measuring greater than 3x the width of the cell body). n=3 individual experiments in triplicate. Means +/−SEM. C-D, Representative cells after 3 weeks of DMSO vehicle control (C) or 5 nM rotenone (D).
Figure 7
Figure 7
Differentiated PC6-3 cells were treated with rotenone (5 nM) or DMSO vehicle for 7,14, or 21 days. A, Control results testing the effect of overexpression of fission protein Drp1 or the dominant-negative form (dnDrp1) alone on neuritic processes. Overexpression of dnDrp1 alone does not affect neurite length after 3wk. *Drp1 overexpression significantly different than control (p=0.0007) at 3 weeks. B, Overexpression of fission inhibitor dnDrp1 protects against the rotenone-induced loss of processes. n=3 individual experiments, +/−SEM; *Significantly different from respective DMSO control, (ANOVA with Bonferroni post-hoc correction); **Not different than DMSO-vector control; p=0.00167 compared to 5 nM rotenone-vector and p=0.0013 compared to 5 nM-Drp1. Note (A) and (B) from same experimental sets with same controls, grouped separately for ease of visualization.
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