MitoDendra, a fluorescent protein for live imaging of mitochondrial dynamics
In order to develop appropriate imaging tools for live imaging of mitochondrial fusion and transport in motor neurons, the photo-switchable fluorescent protein Dendra (Gurskaya et al., 2006
) was targeted to the mitochondrial matrix (mitoDendra) by adding a cleavable N-terminal pre-sequence derived from subunit VIII of cytochrome c oxidase (COX VIII). A linker peptide was introduced between the pre-sequence and Dendra to improve fusion protein cleavage (). MitoDendra was transfected into primary embryonic rat neurons, and the mitochondrial localization of mitoDendra was confirmed by immuno-staining and colocalization with the mitochondrial proteins MnSOD and cytochrome c ().
Figure 1 Study of mitochondrial dynamics in motor neurons using the photo-activatable fluorescent protein Dendra. A, Schematic representation of the mitoDendra construct used to label mitochondria. Restrictions sites are indicated. B, Co-staining with MnSOD and (more ...)
First, we determined the optimal confocal microscopy imaging conditions to visualize and photo-activate mitoDendra in living neurons. Low power 488 nm laser excitation (below 0.5 mW) did not cause changes in the morphology of the transfected neurons or their mitochondria (not shown). Furthermore, there was no photo-conversion to red (, left panels), even after repetitive imaging for extended periods of time (250 times over 2h, data not shown). However, exposure to 10–30 iterations at higher 488 nm laser intensity (2.5 mW) was sufficient to completely and irreversibly photo-convert mitoDendra from green to red fluorescence at a defined region of interest (ROI) (, right panels).
Then, we demonstrated the effectiveness of mitoDendra in studying the dynamics of mitochondria in primary neurons by time-lapse in vivo imaging (). The ability to generate two differentially labeled (green or red) populations of mitochondria within the same neuron by photo-conversion of mitoDendra, allowed for detection of mitochondrial fusion events. In the example shown in , the neuronal mitochondrial network underwent extensive fusion, as demonstrated by the mixing of red and green fluorescence (). We also determined that repetitive imaging did not cause photo-bleaching of mitoDendra fluorescence ().
Impaired mitochondria fusion in G93A SOD1 motor neurons
Mitochondrial fusion is thought to be essential for neuronal survival, since genetic defects of components of the fusion machinery, such as Opa1 and Mfn2, result in neurodegeneration (Chen and Chan, 2006
; Chen et al., 2007
). To determine whether mitochondrial fusion is affected by mutant SOD1 we analyzed the reshaping of mitochondrial network in the soma and axons of motor neurons.
Primary motor neurons from the spinal cords of transgenic rat E14.5 embryos expressing either wild type (WT) or G93A mutant SOD1, and respective non-transgenic embryos, were isolated and transfected with mitoDendra after 5–7 days in vitro (div) for mitochondrial imaging.
In the soma, it was difficult to follow the fate of individual mitochondria, because of the high density of the mitochondrial network, which does not allow for single mitochondria resolution. Therefore, taking advantage of the biophysical properties of mitoDendra, we photo-converted subpopulations of mitochondria in motor neuron cell bodies from green to red fluorescence, and followed their fate by time-lapse confocal microscopy. As a result of the mixing of green and red fluorescence by fusion, yellow mitochondria were detectable in the merged time-lapse images of non-transgenic neurons 40 min after photo-conversion and increased over time (, top panels). However, in G93A SOD1 motor neuron somas, yellow fluorescence was not apparent until 100 min after photo-activation (, bottom panels). Quantification of the fusion rates (colocalization of red over green fluorescence) demonstrated a linear increase (correlation coefficient, r ≥ 0.99) in the amount of colocalization over time, in both control and mutant SOD1 somas, but the rate of fusion was slower (P = 0.013 by ANOVA with repeated measurements; n = 7–8) in G93A motor neuron somas ().
Figure 2 Impaired mitochondrial fusion in the soma and motor axons of G93A SOD1 motor neurons. A, Cell bodies from non-transgenic control and G93A SOD1 motor neurons containing mitoDendra-labeled mitochondria before (−1 min) and after (0 min) photo-activation. (more ...)
In the motor axons, identified by morphological criteria, fusion events involving individual mitochondria could be visualized during time-lapse recordings without applying photo-activation (, insets). Quantification of fusion events involving mobile mitochondria revealed that G93A SOD1, but not WT SOD1, axons had less mitochondrial fusion than non-transgenic controls (P = 0.03 by Student’s t test; n = 16–28). The fusion impairment affected anterograde (P = 0.02 by Student’s t test; n = 12–26), but not retrograde, moving axonal mitochondria ().
Taken together these results indicate that in G93A SOD1 neurons there is a defect of mitochondrial dynamics, which involves fusion and affects both axons and cell bodies.
Defective transport of mitochondria in G93A SOD1 motor neurons
Mitochondrial fusion defects in neurons can be accompanied by impaired mitochondrial motility, although whether the two defects are mechanistically linked or if they can develop independently is still unclear (Misko et al., 2010
). Therefore, we investigated mitochondrial transport dynamics in mutant SOD1 motor neurons. The ability to discriminate between different populations of mitochondria within the same cell using mitoDendra photo-activation was used to study mitochondrial transport in the motor neuron somas. We followed the transport of green mitochondria into the photo-converted (red) areas () and, vice versa, the transport of red mitochondria into the non-photo-converted (green) areas (not shown) over time. We found that the rate of transport of both green () and red () mitochondria within the cell soma was reduced in G93A motor neurons, as compared to non-transgenic controls (P
= 0.011 and P
= 0.039, respectively, by Student’s t test at 100 min; P
= 0.025 and P
= 0.032, respectively, by ANOVA with repeated measurements; n = 7–8).
To quantify fast axonal transport of individual mitochondria in the axons of motor neurons we generated kymographs from time-lapse recordings. Kymographs provide graphical representations of spatial position over time, where the y axis represents time and the x axis, distance. A decrease in the number of diagonal traces indicated a reduction in the number of moving mitochondria in G93A axons (). Mitochondrial motility, defined as the proportion of mitochondria moving in any direction during the time of the recording (5 min), was decreased in G93A SOD1 motor neurons (17.23 ± 2.29 %) as compared to non-transgenic controls (29.01 ± 2.36 %; P = 0.0008 by Student’s t test; n = 18–29), while the number of stationary mitochondria in G93A SOD1 motor neurons was increased (82.77 ± 2.29 % mutant SOD1 versus 70.99 ± 2.36 % non-transgenic controls; P = 0.0008 by Student’s t test), consistently with the decreased motility. Motility in WT SOD1 motor neurons was not significantly different from non-transgenic controls.
Figure 3 Mitochondrial dynamics abnormalities in mutant SOD1 motor neuron axons. A, Representative kymographs of mitoDendra-labeled mitochondria recorded in distal axons from non-transgenic, WT SOD1, and G93A SOD1 motor neurons (MN) at 5 sec intervals for 5 min (more ...)
In order to study if mitochondrial transport abnormalities were widespread along the motor axons or limited to specific axonal regions, we defined two regions: proximal (starting within 20 μm from the cell body and spanning 152 ± 34μm; n = 45 axons), and distal (ending within 20 μm from the axon terminal and spanning 156 ± 41 μm; n = 36). By focusing the analysis on proximal or distal segments of the motor neuron axons and on the direction of the movement, we identified a selective decrease of retrograde transport, in both proximal and distal regions of G93A SOD1 (P = 0.013 and 0.017, respectively, by Student’s t test; n = 6–17), but not in WT SOD1 motor neurons (), as compared to non-transgenic controls.
To further characterize the effects of mutant SOD1 on mitochondrial transport, we analyzed the frequency and duration of pauses, the velocity of transport, the distance traveled between pauses, and the persistence (duration) of movement. We observed that mitochondria paused more often in the retrograde direction in mutant SOD1 motor neurons (; P = 0.016 by Student’s t test; n = 18–27 axons), while there were no significant differences in the duration of the pauses (). Moreover, the velocities in the retrograde direction were reduced in mutant motor neurons (; P= 0.04 by Student’s t test; n = 123–355 moving events). Since there were no significant differences in the persistence of movement of mitochondria (), the distance traveled by mobile mitochondria was shorter in G93A motor neurons (; P = 0.048 by Student’s t test; n = 56–128 moving events). Finally, we observed an imbalance in the distance traveled by mitochondria in distal segments of G93A SOD1 axons: retrograde distances were shorter (P = 0.033 by Student’s t test; n = 59–118 moving events), whereas anterograde distances were longer (; P = 0.013 by Student’s t test; n = 59–118 moving events).
To determine whether fast axonal transport defects affected other cargos we labeled membrane bound organelles (MBO) with amyloid precursor protein (APP)-Dendra () (Koo et al., 1990
; Kaether et al., 2000
). Kymographs of APPDendra labeled MBO demonstrated higher motility of these vesicles () compared to mitochondria (70 ± 4.0 % of MBO in control motor neurons moved within the time-frame of the recording, compared to approximately 30 % of mitochondria). Unlike mitochondria, the anterograde and retrograde motility of MBO was unchanged in proximal and distant segments of G93A SOD1 motor neurons (). Velocity of MBO transport was also unaffected (), in both directions and axonal segments. These results suggest that in motor neurons the fast axonal transport of mitochondria, but not that of other fast moving organelles, such as MBO, is affected by mutant SOD1.
Figure 4 No abnormalities in the transport of membrane-bound organelles (MBO). A, Schematic representation of the APPDendra construct used to label MBO. B, Representative kymographs of APPDendra-labeled MBO recorded from control and G93A SOD1 motor neurons at (more ...)
Reduced mitochondrial length and density in G93A SOD1 motor neurons
A logical consequence of impaired mitochondrial transport and fusion could be the disruption of normal mitochondrial morphology and distribution along axonal processes. Therefore, to first assess mitochondrial morphology we measured the size of mitochondria in proximal and distal regions of motor neuron axons. Mitochondrial average length was reduced in the distal segments of G93A SOD1 motor neuron axons, but not in WT SOD1 (), as compared to non-transgenic controls (P = 0.05−10 by Student’s t test; n = 192–331 mitochondria). Furthermore, while at 5 div mitochondria in the proximal segments of G93A SOD1 motor axons were of normal size (), at 10 div their length had significantly decreased as compared to controls (3.33 ± 0.24 % mutant SOD1 versus 4.03 ± 0.24 % non-transgenic controls; P = 0.04 by Student’s t test; n = 113–138), suggesting that these abnormalities progress from distal to proximal segments as the neurons age in culture. No significant decrease in mitochondrial length was detected in dendrites of mutant SOD1 motor neurons (), indicating that mitochondrial morphological abnormalities were limited to motor axons.
Figure 5 Mitochondrial morphological abnormalities in axons, but not dendrites, of mutant SOD1 motor neurons. A, Images of mitoDendra-labeled mitochondria in distal axonal segments of non-transgenic, WT SOD1, and G93A SOD1 motor neurons (MN). Scale bar, 5 μm. (more ...)
Second, to assess mitochondrial distribution in motor neuron axons we measured the average number of mitochondria contained in proximal and distal axonal segments. Despite the alterations in retrograde transport, there were no significant differences, either in proximal or distal segments, in the number of mitochondria among mutant SOD1, WT, and non-transgenic control axons (). We confirmed this lack of changes in mitochondrial distribution in G93A SOD1 motor neurons by labeling mitochondria with Mitotracker and cytochrome c antibodies (data not shown). Moreover, the distribution of mitochondria at the axon terminals, assessed by fluorescence (, top panels) and electron microscopy (, bottom panels) did not appear altered in G93A SOD1 motor neurons. Further quantification of mitochondrial distribution in defined 25 μm segments did not reveal significant differences among groups at the axonal terminals ().
We then analyzed mitochondrial “density”, expressed as the sum of all the mitochondrial lengths in 50 μm axonal segments. Mitochondrial density was reduced in the distal segments of G93A SOD1 axons, but not WT SOD1, as compared to non-transgenic controls ( = 0.027 by Student’s t test; n = 23–29 axons). However, no differences were observed between control and mutant SOD1 when measuring the density of mitochondria in the motor neuron cell bodies (data not shown).
These results suggest that mutant SOD1 may cause an imbalance in the distribution of mitochondria along the axons, which appears first at the periphery of the motor neurons. Although the overall number of mitochondria was unchanged in mutant SOD1 and control axons, there was a decrease of mitochondrial density, as measured by mass, in the mutants due to smaller average mitochondrial size.
Mitochondrial dynamics and morphology are not altered in G93A SOD1 cortical neurons
To investigate if abnormalities in mitochondrial dynamics and morphology are specific to motor neurons or they affect other neuronal types, we studied cortical neurons from G93A SOD1 and control rat embryos at 8 div. Kymographs revealed a similar motility pattern for mitochondria in both non-transgenic controls and G93A SOD1 cortical neurons (). After analysis of time-lapse recordings, no significant differences in the frequency of mitochondrial movement were found in either proximal or distal segments of the axons (), and in either anterograde or retrograde directions (not shown).
Figure 6 No abnormalities in transport or morphology of mitochondria in mutant SOD1 cortical neurons. A, Representative kymographs from proximal segments of non-transgenic and G93A SOD1 cortical neurons at 8 div. Color in the image was inverted (negative image) (more ...)
The average mitochondrial length in both proximal and distal axonal segments was also unchanged in G93A SOD1 cortical neurons at 8 div (), and no differences in the number of mitochondria and mitochondrial density were found (not shown).
To exclude that abnormalities developed in cortical neurons at later time points, we analyzed mitochondrial frequency of transport and morphological parameters in proximal and distal segments of neurons at 15 div, and confirmed that there were no changes in G93A SOD1, as compared to non-transgenic controls (not shown).
We then compared SOD1 expression levels in purified cortical neurons and spinal motor neurons by Western blot (). The relative amount of G93A SOD1 in purified cortical neurons was higher than in motor neurons (), excluding that the lack of mitochondrial abnormalities in cortical neurons was related to lower mutant protein load.
Accumulation of dysfunctional mitochondria in distal regions of mutant SOD1 motor neurons
Abnormalities of mitochondrial fusion and transport in mutant SOD1 motor neurons could be linked, either as a cause or as a consequence, to mitochondrial functional impairment. Thus, to explore whether there is a functional correlate to the abnormalities in mitochondrial dynamics and morphology in G93A SOD1 motor axons we investigated mitochondrial membrane potential (ΔΨ). Motor neurons were loaded with the potentiometric fluorescent dye TMRM (5nM) and the intensity of fluorescence was used as a relative measure of ΔΨ (Vives-Bauza et al., 2008
). We observed reduced average TMRM intensity in the mitochondria in distal segments of mutant SOD1 motor neurons ( = 0.001 by Student’s t test; n = 97–145), suggesting that bioenergetic dysfunction in distal motor axon segments may be correlated with reduced size and impaired dynamics of mitochondria. There was a slight but statistically significant increase of TMRM intensity of mutant SOD1 mitochondria in proximal segments ( = 0.022 by Student’s t test; n = 110–140). The reasons for this increase are not clear, but it could be due to the increase of the ratio of stationary over retrograde moving mitochondria, which are thought to have lower ΔΨ than stationary ones, in neurons (Miller and Sheetz, 2004
Figure 7 Impairment of mitochondrial bioenergetics in mutant SOD1 motor neuron axons. A, Average TMRM fluorescence intensity (mitochondrial membrane potential) of all mitochondria in proximal and distal segments of non-transgenic and G93A SOD1 motor neurons (8 (more ...)
Next, TMRM labeled mitochondria were imaged by time-lapse microscopy, and kymographs were obtained in order to simultaneously assess mitochondrial transport and ΔΨ. For each genotype, the average TMRM intensities measured for mobile or stationary mitochondria were expressed relative to the average TMRM intensity of all mitochondria analyzed (set at 1). The kymographs confirmed the reduced motility of mitochondria in distal regions of mutant SOD1 motor neurons (). Anterograde-moving mitochondria in mutant SOD1 motor neurons had lower ΔΨ than anterograde-moving mitochondria in controls (P = 0.0017 by Student’s t test; n = 30–37), and were similar to retrograde-moving mitochondria in both mutant and control neurons (). This finding has potential pathogenic significance, because it suggests that the pool of upcoming mitochondria destined to replace or fuse with distal aging mitochondria in mutant SOD1 motor axons may be functionally impaired.
Fewer and smaller synaptic puncta in G93A SOD1 motor neurons
Since neuronal mitochondria are often localized in proximity of synapses to provide the necessary energy and calcium buffering, we hypothesized that impaired mitochondrial transport in mutant SOD1 motor neurons could be associated with synaptic abnormalities. To investigate mitochondrial localization at synaptic sites we looked at the synaptic contacts that are spontaneously formed among motor neurons in vitro, and used these structures as reference sites, where to study mitochondrial localization. We labeled the neuron-to-neuron synapses with antibodies against the pre-synaptic proteins SV2 and synapsin I, and measured both the number and the size of synaptic puncta. G93A SOD1 motor neurons presented a markedly decreased number of SV2- and synapsin I-positive puncta (P = 0.05−11 and P = 0.000004 by Student’s t test for SV2 and synapsin I labeling, respectively; n = 21–27 axons) and smaller puncta sizes (P = 0.0006 and P = 0.00011 by Student’s t test for SV2 and synapsin I labeling, respectively; n = 190–385 puncta) as compared to controls ().
Figure 8 Synaptic abnormalities in G93A SOD1 motor neurons. A, Representative images of synaptic structures labeled with SV2 and synapsin I in control and G93A SOD1 motor neurons. Scale bar, 5 μm. B, Analysis of puncta density (number of puncta per 50 (more ...)
To determine whether abnormal mitochondrial distribution was associated with the observed synaptic abnormalities, we measured the colocalization of mitochondria and SV2-labeled puncta (). Two parameters were utilized for the measurements: the first was the probability of finding a mitochondrion localized within 1 μm from the puncta; the second was the mitochondrial density at the puncta, defined as the sum of all the mitochondrial lengths within 5 μm from each puncta. Both the probability of mitochondria-puncta colocalization () and the puncta mitochondrial density () were reduced in the axons of G93A SOD1 motor neurons (P = 0.018 and P= 0.010 by Student’s t test, respectively; n = 20–22 axons)
In order to focus on functional synapses and to investigate differences in synaptic function, we used the dye AM4-65. AM4-65 is loaded into synaptic vesicles at functional synapses under synaptic activation (Cochilla et al., 1999
). We analyzed axonal segments from mutant SOD1 and control motor neurons after incubation with AM4-65 under depolarizing conditions. Quantification of the density of AM4-65 puncta revealed a reduction in the number of synaptic puncta in G93A SOD1 motor neurons (P
= 0.000007 by Student’s t test; n = 20–29 axons; ), which indicates that the synaptic morphological changes correlated with synaptic dysfunction.
These findings suggest the possibility that a lack of appropriate supply of functional mitochondria is involved in causing synaptic abnormalities in mutant SOD1 motor neuron axons.