Quantified, direct observation of mitochondrial fission and fusion (DrOF) in neurons can be used in chronic neuronal models
We first established long-term primary neuronal cultures and utilized our previously established methodology for quantitatively assessing mitochondrial fission and fusion using fluorescent and photoactivatable mitochondrially targeted proteins, direct evaluation of mitochondrial fission and fusion (DrOF) (Berman et al., 2009
), as well as methodology for evaluating mitochondrial transport in primary neurons. Earlier work utilized primary rat cortical neurons that were DIV 8 (Berman et al., 2009
), and we found that we were able to utilize DrOF to analyze mitochondrial fission and fusion in neurons even after four weeks (28 DIV; 22 days after transfection). An example in a single neuronal process, in which a photoactivated (yellow) mitochondrion undergoes fusion with a non-photoactivated mitochondrion containing mtDsRed2, followed by a fission event is shown in and Supplemental Movie 1
. Using this methodology, we are able to distinguish true fusion events from passing mitochondria, which occurs much more frequently than fusion (Berman et al., 2009
). One caveat to these studies is that we could not distinguish between axons and dendrites, which could be an important distinction if presynaptic versus postsynaptic mechanisms are involved in the neuropathologic process. However, although there is some evidence that mitochondrial morphology and distribution can differ between axons and dendrites (Chang et al., 2006
), differences in rates of fission and fusion have not been studied, and mitochondrial transport has been reported to be similar between axons and dendrites (Ligon and Steward, 2000
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 (more ...)
Age-related changes in mitochondrial fission and fusion
We found that mitochondrial fission and fusion rates change as neurons mature/age in culture. Fission rates were higher in neurites of DIV 7 neurons than in older neurons (DIV 28; ). Fusion rates, however, remained unchanged. This suggests that higher rates of mitochondrial fission occur at the time period while synaptic connections are being formed (Chang and Reynolds, 2006
), and a lower rate as neurons age in culture, leading to an overall decrease in the ratio of fission to fusion. Although this is clearly not an ideal model of ‘aging’, there is evidence suggesting that primary neurons in culture do show signs suggestive of senescence by this age (Blalock et al., 1999
; Chernova et al., 2006
; Xiong et al., 2004
). Thus, this could reflect physiologic aging-associated changes in mitochondrial fission and fusion.
Chronic rotenone-induced effects on mitochondrial fission and fusion change over time
We then developed a chronic neuronal model to look at changes in mitochondrial dynamics over time, prior
to cell death. In Parkinson’s disease (PD), mitochondrial fission had been implicated in high-concentration, acutely
lethal rotenone toxicity, but whether this is relevant to the chronic, slow changes in neurodegenerative disease is unclear. Therefore, we modified the well-established model of chronic rotenone, in which chronic, systemic rotenone in rats results in specific pathology similar to that seen in human PD (Betarbet et al., 2000
), in order to evaluate a potential role for mitochondrial dynamics in early phases of the neurodegeneration of Parkinson’s disease (PD). We first utilized a low-concentration, chronic rotenone treatment model in cell culture and used primary rat neurons to look directly at mitochondrial dynamics after chronic rotenone treatment. We utilized DrOF and direct manual tracking to provide direct evaluation of the effect of chronic low-dose rotenone on mitochondrial dynamics in living neurons.
We wanted to develop a model in which the concentration of chronic rotenone did not lead acutely to a large amount of cell death, but rather was able to be provided over several weeks, in order to more closely model the chronic condition of PD. In these studies, then, we utilized primary rat cortical neurons, began treatment with 0.1 nM or 1 nM rotenone or vehicle control at 7 DIV, and treated for one and two weeks duration. At these low concentrations of rotenone, there was little increase in cell death, whereas higher concentrations of rotenone did cause significant neuronal cell death (Supplemental Figure 1A
). In addition, these neurons were able to maintain normal ATP levels (Supplemental Figure 1B
). This is consistent with previous data in cell culture that show that concentrations of rotenone less than 15 nM had minimal effect on mitochondrial respiration (Sherer et al., 2002
Although up to two weeks of exposure to low concentrations of rotenone caused little cell death, we discovered effects on mitochondrial dynamics, resulting in quantitative changes in fission and fusion. We observed that one week of exposure to our higher concentration of rotenone (1 nM) resulted in a significant increase in mitochondrial fusion (), an effect that was gone by two weeks of exposure. The lower concentration of rotenone did not have significant effects on fusion itself, though analysis is limited by the rarity of fusion events. Conversely, after two weeks of exposure to low-dose rotenone (0.1 nM), mitochondrial fission increased significantly (). Based on these data, one can estimate the overall ratio of fission events to fusion events. This reveals that after one week of exposure to 1 nM chronic rotenone, the ratio of mitochondrial fission to fusion in neurons decreases, secondary to the increase in fusion (). However, after two weeks, the overall ratio of mitochondrial fission to fusion increases in both rotenone conditions compared to vehicle control ().
Chronic rotenone increases the frequency of linked fusion/fission events
Additionally, we observed an interesting phenomenon: chronic exposure to rotenone also increased the number of mitochondria undergoing fusion (with mixing of matrix contents) followed by linked fission (), defined as a fusion event followed by a fission event in the resulting mitochondrion within the 15 minute observation period. These events were rare in general in neuronal processes, but when occurring, the fission events occurred from 60 to 260 seconds following the initial fusion event, with an average duration of 151 seconds between the fusion and fission. This is similar to the time course of linked fusion/fission events noted in non-neuronal cells that was proposed to be involved in autophagic degradation of damaged mitochondria (mitophagy) (Twig et al., 2008
Chronic rotenone alters mitochondrial trafficking
To further evaluate possible interrelated changes in mitochondrial dynamics, we then quantitatively evaluated the effects of low-dose, chronic rotenone on neuronal mitochondrial anterograde and retrograde transport. Using live imaging of primary cortical neurons transfected with mtDsRed2, treated under control or chronic rotenone conditions, images were taken every 10 s at 37° for 15 min as described above for fusion/fission analysis. Using manual object tracking software (as described in Methods
), we were able to individually track each fluorescent mitochondrion in a given neurite at each 10s imaged timepoint, as well as measure its starting length. Excel macros were developed to calculate average and maximal velocities, as well as direction from the neuron cell body (anterograde or retrograde) ( and Supplemental Movie 2
). While we could not reliably distinguish between axons and dendrites in this living system, there is evidence that mitochondrial transport is similar between them (Ligon and Steward, 2000
Figure 2 Mitochondrial transport after chronic rotenone. Cortical neurons transfected and treated with chronic rotenone as in were imaged every 10 s for 15 min. A-C, Example of manual tracking using Image J with Manual Tracking plug-in as described. (more ...)
After two weeks of exposure to 0.1 nM rotenone, changes in mitochondrial transport occurred. Surprisingly, we found that overall average velocity increased when neurons were exposed to a very low concentration of rotenone (0.1 nM; ), but not 1 nM rotenone. Both concentrations of rotenone resulted in a higher proportion of retrograde mitochondrial transport (). Using this methodology, we were also able to evaluate the mitochondrial length/velocity distribution of the mitochondrial in the neuronal processes (). This analysis suggests that an increase in the population of very small, fast moving mitochondria contribute to the higher average velocity of the rotenone-treated neurons as compared to controls. One can observe that this increase in fast-moving, small mitochondria appears to start earlier in neurons exposed to the higher concentration of rotenone (2F) and takes longer to develop in the lower concentration of chronic rotenone (2G). Earlier in exposure (1 week), neuronal mitochondria did not show a significant difference in mitochondrial velocity (not shown) but begin to show a similar distribution of smaller, fast-moving mitochondria at higher concentrations of rotenone. Although we did not directly quantify this, our observations suggest that the increased velocity is due to a higher proportion of mitochondria undergoing fast transport with minimal ‘stopping’ than to an increase in speed of transport.
Chronic rotenone leads only to minor changes in mitochondrial morphology in neurons
In order to further explore the implications of the changes in mitochondrial fission and fusion observed after chronic rotenone in neurons, we evaluated mitochondrial morphology in the neuritic processes. In contrast to what might be predicted by a simple model of mitochondrial morphology being related to the simple balance of fission and fusion, where we might expect the increased mitochondrial fission and fission/fusion ratio after two weeks of chronic rotenone () to result in smaller, more fragmented mitochondria, we found that we did not see fragmentation of mitochondria. Instead, when mitochondrial fission was greatest (2 week, 0.1 nM rotenone, ), mitochondrial length was unchanged (). Although the fission/fusion ratio increased also after 2 weeks of 1 nM rotenone exposure (), average mitochondrial length actually increased (). We thus began to explore possible mechanisms for this apparent conundrum.
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 (more ...)
Chronic rotenone does not cause preferential directional transport of differently sized mitochondria
One explanation for longer or unchanged morphology in mitochondria in neuritic processes, despite increases in mitochondrial fission in processes, would be that longer cell body mitochondria are transported away from the cell body to replace the smaller mitochondria in neurites. Alternatively, smaller mitochondria in processes could be transported back to the cell body. This was of particular interest, given the potential role for targeting damaged mitochondria for mitophagy and the role that linked fusion/fission events might play. One might suspect that fission results in division into damaged, smaller mitochondrial components that could be transported back towards the cell body for degradation, especially since we had observed an increase in smaller, faster-moving mitochondria and in retrograde movement after rotenone (). Therefore, we evaluated the size of mitochondria being transported away from and towards the cell body. However, we found no evidence for preferential transport based on mitochondrial size. We found no significant difference in the size of the anterogradely or retrogradely moving mitochondria in any conditions (). The slightly longer mitochondria noted in the processes after two weeks of the higher concentration of rotenone cannot, then, be explained by preferential directional transport of certain-sized mitochondria.
Chronic rotenone increases mitochondrial density in distal neuronal processes prior to changes in cell bodies
We also asked whether one possible mechanism for longer mitochondria in neuronal processes might be increased mitochondrial growth or biogenesis. If mitochondria were lengthened out in the distal process, followed by mitochondrial fission to make two daughter mitochondria, this might explain the greater rate of fission along with longer mitochondria. Alternatively, if the increase in fusion/fission events were linked to mitochondrial degradation through mitophagy, we might expect to see less mitochondrial density in neuritic processes. As a first pass to begin to look at these factors in compartmentalized regions of neurons, we evaluated mitochondrial density in neuronal cell bodies, proximal processes (approximately 300 uM from cell body) and distal processes (approximately 1200 um from cell body) (). Example neurons are shown in . We noted that as neurons age in vitro, mitochondrial density increases under DMSO vehicle control conditions (). In addition, we found evidence of differential changes in different regions of the neurons in response to chronic toxicity. After one week of rotenone treatment, we found no change in overall mitochondrial density in neuronal cell bodies compared to control conditions (as estimated by total mitochondrial volume as a proportion of total cell body volume; ). However, out in neuronal processes, changes do occur. We found greater mitochondrial density in the distal processes after one week of rotenone treatment, but no significant change in the proximal processes. After two weeks, we see even greater increases in mitochondrial density in the distal processes after chronic rotenone (1 nM) treatment compared to control (). Interestingly, after two weeks of rotenone treatment, we now observe a significant increase in mitochondrial volume in the cell body as well. This is the same time point when longer mitochondria in processes are noted. This suggests the possibility that chronic, compensatory changes occur, possibly increased biogenesis or growth, in an attempt to compensate for accumulating mitochondrial toxicity over time. On the other hand, there is no evidence of loss of mitochondria after chronic rotenone treatment. This also supports that mitochondrial dynamics can be differentially affected over time in specific subcellular regions.
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 (more ...)
Chronic low-concentration rotenone does not alter autophagy nor affect mitophagy in neurons
To corroborate our findings with regard to mitochondrial density, we more directly evaluated autophagy and specific mitochondrial autophagy (or mitophagy), which has been hypothesized to be an important mechanism of clearing dysfunctional mitochondria (Tolkovsky, 2009
), and which has been linked to the function of the PD-linked gene products parkin and PINK1(Dagda et al., 2009
; Geisler et al., 2010
; Kawajiri et al., 2010
; Narendra et al., 2008
; Narendra et al., 2010
; Vives-Bauza et al., 2010
). We wondered whether autophagic clearance was altered and were specifically interested in whether autophagy of mitochondria might be altered, and whether this might contribute to the density changes noted. Utilizing immunocytochemistry to assess whether accumulations of the autophagic vesicle marker LC3 were present under our conditions, we found that approximately 20-25% of neurons contained more than four autophagic puncta under control conditions, and this was unchanged by chronic rotenone (). We only rarely detected autophagic puncta in distal neurites under any conditions (not shown). Using endogenous LC3 immunostaining along with mtDsRed2 to mark mitochondria, we also attempted to evaluate whether specific mitophagy in neurons was altered after chronic rotenone. Although we could occasionally detect LC3-containing vesicles surrounding mitochondria in neurites and cell bodies, and when coexpressing GFP-tagged LC3 in order to perform live imaging, we could occasionally detect these mitochondria-containing vesicles in neurites transported towards cell bodies (not shown), these were detected very rarely, and the vast majority of neurons under any conditions did not reveal significant colocalization with LC3 and mitochondrial fluorescence. This was true equally in control and rotenone-treated conditions (), suggesting that at least in our model system, we could not detect a role for mitophagy, though low-level changes cannot be ruled out.
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 (more ...)
Chronic low-concentration rotenone results in loss of neuronal processes without cell death in dopaminergic neuronal cells
We now wanted to know whether these early changes in mitochondrial dynamics could be important in neuropathology. To begin to examine this, we wanted to utilize a dopaminergic neuronal model, so we modified the chronic rotenone model to nerve growth factor (NGF)-differentiated, dopamine-producing PC6-3 cells. After seven days of differentiation, standard media was changed to media containing either DMSO vehicle control or concentrations of rotenone ranging from 1 nM to 100 nM, for one, two, or three additional weeks (). We found that at concentrations under 25 nM, little cell death occurred, even after 3 weeks of exposure (). Interestingly, however, we found that in these dopaminergic cells, the low concentrations of chronic rotenone did
result in clear neuropathologic changes that have been observed both in PD and in the systemic chronic rotenone animal model of PD (Betarbet et al., 2000
)), specifically that these differentiated cells began to lose their neuritic processes, although they do not die (), similar to that observed in SH-SY5Y cells (Borland et al., 2008
). We took this loss of neurites to be indicative of early pathology that could be similar in nature to that seen in PD and other PD models. Mitochondria from processes after chronic low-dose rotenone, however, were indistinguishable in general morphology from vehicle-treated controls (not shown). This is in contrast to the dramatic fragmentation reported after acute rotenone treatment in other cell types (Barsoum et al., 2006
), suggesting again significant mechanistic differences between acute high-dose and lower-dose chronic models.
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 (more ...)
Reducing balance of mitochondrial fission protects against loss of neurites
We then asked whether the altered mitochondrial dynamics might be influencing toxicity at this early stage. One could hypothesize that reducing the overall balance of fission could be either protective or harmful to neurons. If excessive fission is promoting cell death, or if fusion is critical for maintaining healthy mitochondria exposed to chronic rotenone, reducing fission could be protective. However, if the role of fission in maintenance of synapses and mitochondrial distribution is important, then promoting fission could be protective to early neuropathology.
To begin to evaluate how mitochondrial dynamics contribute to the early neuropathology of neuritic processes, we thus examined the effects of overexpression of the mitochondrial fission protein Drp1 or its dominant negative form, mutant Drp1K38A
(Labrousse et al., 1999
; Smirnova et al., 1998
) on these early changes in neuronal process morphology. We have previously shown directly in neurons that overexpression of the dominant-negative Drp1K38A
resulted in a shift towards increased mitochondrial fusion in neurons (Berman et al., 2009
). In neuronally-differentiated dopaminergic PC6-3 cells treated for three weeks with low-concentration rotenone, we found that Drp1K38A
had no effect on neuritic processes under control conditions (), although unexpectedly, overexpression of Drp1 itself resulted in a loss of neuronal processes, regardless of whether rotenone was present or not. However, we also found that overexpression of Drp1K38A
resulted in significant protection against the rotenone-induced loss of neuritic processes compared with vector-transfected control cells (). The effect was greatest after 3 weeks of chronic rotenone treatment.
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 (more ...)