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Mutations in Cu,Zn superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis (FALS), a rapidly fatal motor neuron disease. Mutant SOD1 has pleiotropic toxic effects on motor neurons, among which mitochondrial dysfunction has been proposed as one of the contributing factors in motor neuron demise. Mitochondria are highly dynamic in neurons; they are constantly reshaped by fusion and move along neurites to localize at sites of high-energy utilization, such as synapses. The finding of abnormal mitochondria accumulation in neuromuscular junctions, where the SOD1-FALS degenerative process is though to initiate, suggests that impaired mitochondrial dynamics in motor neurons may be involved in pathogenesis. We addressed this hypothesis by live imaging microscopy of photo-switchable fluorescent mitoDendra in transgenic rat motor neurons expressing mutant or wild type human SOD1. We demonstrate that mutant SOD1 motor neurons have impaired mitochondrial fusion in axons and cell bodies. Mitochondria also display selective impairment of retrograde axonal transport, with reduced frequency and velocity of movements. Fusion and transport defects are associated with smaller mitochondrial size, decreased mitochondrial density, and defective mitochondrial membrane potential. Furthermore, mislocalization of mitochondria at synapses among motor neurons, in vitro, correlates with abnormal synaptic number, structure, and function. Dynamics abnormalities are specific to mutant SOD1 motor neuron mitochondria, since they are absent in wild type SOD1 motor neurons, they do not involve other organelles, and they are not found in cortical neurons. Taken together, these results suggest that impaired mitochondrial dynamics may contribute to the selective degeneration of motor neurons in SOD1-FALS.
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting motor neurons. The disease begins in mid-life and is rapidly progressive, leading to paralysis and death within a few years of onset (Rowland and Shneider, 2001). While the majority of the cases of ALS are sporadic with unknown etiology, about 10% are inherited (familial ALS, FALS), of which approximately 20% have mutations in the Cu,Zn superoxide dismutase gene (SOD1) (Rosen et al., 1994).
There are several, interconnected, pathogenic pathways leading to SOD1-FALS, since mutant SOD1 has a multitude of pathogenic effects in neurons and in surrounding cells, such as astrocytes and microglia (Ilieva et al., 2009). Among the several proposed mechanisms of toxicity, which include protein aggregation, neuroinflammation, excitotoxicity, oxidative stress, growth factor deficiency, glial pathology, cytoskeletal alteration, and axonal transport abnormalities (Rothstein, 2009), mitochondrial defects are thought to play a contributing role (Shi et al., 2010; Magrane and Manfredi, 2009). The toxic effects of mutant SOD1 on mitochondria occur at multiple levels, affecting their bioenergetics (Mattiazzi et al., 2002; Damiano et al., 2006), their channel properties (Israelson et al., 2010), protein import (Li et al., 2010), and the balance of anti- and pro-apoptotic proteins (Pedrini et al., 2010). In neurons, mitochondria form a highly dynamic and interconnected network that undergoes transport and remodeling by fusion and fission (Chan et al., 2006). Therefore, in mutant SOD1 neurons mitochondrial impairment may also be linked to defects in their dynamics (Magrane and Manfredi, 2009).
A potential consequence of dynamics impairment in neurons is the failure to provide healthy mitochondria to synaptic terminals and other sites of high-energy consumption. SOD1-ALS originates as a distal axonopathy, where neuromuscular junctions (NMJ) are the first regions of the motor neurons to degenerate (Frey et al., 2000; Fischer et al., 2004; Schaefer et al., 2005). Abnormal mitochondria at the NMJ of mutant SOD1 mice appear early on in the course of the disease, and correlate with the beginning of denervation (Vande Velde et al., 2004; Gould et al., 2006), suggesting that a defect in the maintenance of functional mitochondria may be participating in the pathogenic process.
To address the effects of mutant SOD1 on mitochondrial dynamics we investigated motor neurons derived from animal models of SOD1-FALS (G93A mutant and wild type SOD1) using time-lapse confocal microscopy and the photo-switchable fluorescent protein, Dendra (Gurskaya et al., 2006), targeted to mitochondria (mitoDendra). Dendra naturally fluoresces green, but has the distinctive property of changing to red when photo-activated with 488 nm light. This photo-conversion is irreversible, non-toxic, and stable, which makes Dendra an ideal tool for real-time and long-term imaging.
We demonstrated in this study that mitochondrial fusion was decreased and axonal transport impaired in G93A mutant SOD1 motor neurons. These abnormalities correlated with reduced mitochondrial size and density along axons, and with impaired mitochondrial bioenergetics. Changes in mitochondrial dynamics and morphology affected motor neurons, but not cortical neurons. Furthermore, we found that axonal transport defects were specific to mitochondria, since transport of other organelles was intact. Finally, we showed that mitochondrial defects were associated with functional and morphological abnormalities in the synaptic structures that are formed in vitro among motor neurons, suggesting that these defects may play a role in motor neuron synaptic degeneration.
Live imaging experiments were performed in Lab-Tek 4-well chambered glass slides (Nalge Nunc International). The following antibodies were used: cytochrome c (BD Pharmigen), MnSOD (Stressgen), SOD1 (Calbiochem), β-actin (Sigma), SV2 (Developmental Studies Hybridoma Bank), synapsin I (Sigma), and secondary Cy2-, Cy3-, Cy5- and HRP-conjugated IgG (Jackson Immunochemicals). Fluorescent dyes MitoTracker Red CMXROS and tetramethyl rhodamine methyl ester (TMRM) were from Invitrogen.
In the pTurbo-mitoDendra expression vector, Dendra coding region substituted the GFP at the AgeI and NotI sites of the pTurboGFP-mito (Evrogen). In the amyloid precursor protein (APP695)-Dendra expression vector, APP695 cDNA was cloned into pTurbo-DendraV-N using HindIII and AgeI sites. All constructs were verified by sequencing.
Transgenic rats expressing human mutant G93A SOD1 or wild-type (WT) SOD1 were on a Sprague-Dawley genetic background (Chan et al., 1998; Howland et al., 2002). All experiments were performed on neurons derived from at least three independent isolations. Each isolation consisted of motor neurons prepared by mixing all littermate embryos with the same genotype (transgenic or non-transgenic) from one pregnant rat. Genotyping was performed by PCR using the following pair of primers for human SOD1 transgene: IMR 113 (5′-catcagccctaatccatctga) and IMR 114 (5′-cgcgactaacaatcaaagtga). All experiments were approved by the Weill Cornell Medical College Institutional Animal Care and Use Committee.
Purified motor neurons were prepared from E14.5 rat embryo spinal cords, and cultured as previously described (Estevez et al., 1998). Embryos were kept at 4°C in Hybernate E medium (BrainBits) supplemented with B27 (Invitrogen), while genotype analysis was performed. Motor neurons (7,500–10,000 per chamber; 5,000 per cover slip) were plated onto poly-ornithine and laminin coated surfaces (3 μg/ml each). Transgenic and non-transgenic control embryos were from the same litters. Motor neurons were transfected after 5–7 days in vitro (div) with 1 μg cDNA and 1 μl Lipofectamine 2000 (Invitrogen); medium was changed after 4–6 h. Imaging was performed 24–36 h later.
Primary rat cortical neuron cultures were prepared as described (Magrane et al., 2004), and plated onto poly-ornithine and laminin coated surfaces (3 and 5 μg/ml, respectively). Cortical neurons were transfected with mitoDendra after 7 div, and imaged 24–36 h later. For live imaging studies, axons were identified according to morphological criteria (long neurites, constant thin diameter, no branching, perpendicular emergence from the cell body).
The confocal microscope and live imaging station used were previously described (Magrane et al., 2009). The 488 nm laser intensity was kept below 0.5 mW to prevent spontaneous photo-activation of Dendra. A 63x oil immersion lens, and a pinhole of 2.89 AU (optical slice < 2 μm) were applied for mitochondrial imaging; pinhole opening was set to 5.78 AU (optical slice < 4 μm) for membrane-bound organelles (MBO) imaging. Images were collected every 5 sec for 5–7 min. Imaged axonal segments were defined as proximal (starting within 20 μm from the cell body) or distal (ending within 20 μm from the axon terminal).
Photo-activation of mitochondria was achieved in half of the motor neuron soma by exposing mitoDendra to 30–50 iterations using the 488 nm laser (7.5 mW). The entire thickness of the half soma was photo-converted by moving the laser to the first, mid, and last z-positions (1.6 μm intervals).
To study fusion and motility of mitochondria in the motor neuron somas both intact and photo-converted mitochondria pools were simultaneously imaged using 488 (0.25 mW) and 543 nm (0.5 mW) excitation, respectively. Frames were acquired prior (−1 min) and after photo-activation (0, 10, 40, 70, and 100 min). Single optical z-sections were used for the analysis, and the same thresholds were applied over time for each channel. ROI were drawn for both photo-activated and non-photo-activated areas. Colocalization and Integrated Morphometric Analysis applications were from Metamorph software (Universal Imaging Co.).
Colocalization (fusion rate) was measured within the photo-activated area, and expressed as percent of the area of red overlapping green fluorescent mitochondria. Analysis of motility was performed on the green and red channels independently. First, the transport of non-photo-activated (green fluorescent) mitochondria over the photo-activated area (no green mitochondria present) was measured by quantifying the number of pixels appearing over time relative to the total amount of mitochondria present before photo-activation (t = −1). Similarly, the transport of photo-activated (red fluorescent) mitochondria over the non-photo-activated area (no red mitochondria present) was analyzed, and the appearance of pixels relative to the total amount of pixels (green and red) present in each time point was measured.
A mitochondrion was defined as mobile if it changed its position in at least three consecutive frames (taken at 5 sec intervals). Transport of mitochondria and MBO along neuronal processes was analyzed using Metamorph software, as described (Magrane et al., 2009). Briefly, we generated kymographs (a graph of position versus time) and studied several transport parameters for anterograde or retrograde moving mitochondria, in proximal and distal regions of the motor axons. Distance was defined as the distance (in μm) traveled by a mitochondrion in either direction from origin to end of the moving event (i.e., distance traveled between two pauses); only distances longer than 3 μm were analyzed. Persistence of movement was the duration (in sec) of a moving event. Velocity of mitochondria and MBO was measured during sustained movement along the axon. Pauses were defined as absence of movement for more than three consecutive frames in mitochondria that were previously moving during the recording. The frequency of pauses was expressed as the ratio number of pauses / number of moving events; fusion events were not considered as pauses. Duration of pauses (in sec) was also measured. Fusion events were identified when two mitochondria merged and became a single one for more than 3 consecutive frames; data was plotted as the ratio frequency of fusion / moving mitochondria.
Proximal and distal segments of axons (see above for definition) were imaged. Criteria for the analysis of mitochondrial length and density were as previously described (Magrane et al., 2009). Mitochondrial distribution in neurites was expressed as the number of mitochondria per 50 μm of neurite segments. Mitochondrial distribution in distal axons was also measured by binning data into 25 μm segments. To study density of mitochondria in the motor neuron somas, regions of interest (ROI) in single optical z-sections were defined, threshold was set to twice the background fluorescence; mitochondrial density was determined by measuring the percentage of thresholded area in the defined ROI using Metamorph.
Neuron-to-neuron synapses were identified by fixing 9 div motor neurons in 3% PFA and immunolabeling them with antibodies against the pre-synaptic proteins SV2 and synapsin I. A 63x oil immersion lens, standard pinhole of 1 and optical intervals of 0.5 μm were used for confocal imaging; 16-bit, non-saturated, images were acquired. Threshold was set to twice the fluorescence intensity of regions of the neuritic shaft devoid of synapses (background). The number and size of the thresholded puncta were determined by using the integrated morphometric analysis feature in Metamorph. To study the relative distribution of mitochondria around synaptic puncta, cells were labeled with 250 nM MitoTracker Red CMXROS for 10 min at 37°C, followed by several washes with medium, fixation, and immunolabeling of synaptic proteins as described above. Circular ROI with a radius of 1 μm from the synaptic puncta were placed around each identified puncta; mitochondria occupying at least half of the radius of an ROI were considered to be localized in that ROI.
Synaptic function was assed in living motor neurons at 9 div by following the endocytosis of the fixable, activity-dependent fluorescent nerve terminal probe AM4-65 (Biotium, Inc.). Briefly, cells were incubated for 90 sec at room temperature with 4 μM AM4-65, which was prepared in high potassium Ringer’s solution (in mM: NaCl 119, KCl 70, CaCl2 2, MgCl2 2, Hepes pH 7.4 5, and glucose 30) to depolarize the neurons. Motor neurons were washed twice for 4 min in low potassium (2.5 mM) Ringer’s solution containing 1 μM tetrodotoxin (TTX), and then fixed as described above.
The potentiometric fluorescent dye TMRM was used to assess changes in ΔΨm. Briefly, motor neurons (8 div) were loaded with 5 nM TMRM at 37°C. Confocal imaging of TMRM fluorescence was performed using low 543 nm excitation (0.02 mW intensity) to keep fluorescent values within a non-saturated range (0–4095 units). A 63x oil immersion lens and a pinhole of 2.89 AU were used. 12-bit images were taken. Average fluorescence in each mitochondrion was determined after thresholding of the images. The threshold was set to twice the fluorescence from the neuritic shaft in areas devoid of mitochondria to provide adequate background subtraction.
Cortices were dissected and cortical neurons were pelleted from individual rat embryos. The ventral part of spinal cords was dissected away, cords from embryos with the same genotype (non-transgenic or transgenic) were pooled after genotyping, and an enriched motor neuron fraction (purity higher than 80%) was obtained by density centrifugation (bovine serum albumin gradient followed by Optiprep gradient) as previously described (Bishop et al., 2009). Cells pellets were solubilized, electrophoresed in 12% SDS-PAGE gels, and immunoblotted for the indicated proteins, as described (Magrane et al., 2004). Band intensities were quantified by densitometry using Image J software.
Primary motor neurons growing on glass coverslips were fixed at 9 div with a modified Karmovsky’s solution of 2.5% glutaraldehyde, 4% parafomaldehyde and 0.02% picric acid in 0.1 M sodium cacodylate buffer at pH 7.2. After a secondary fixation in 1% osmium tetroxide and 1.5% potassium ferricyanide, samples were dehydrated through a graded ethanol series, and embedded in an epon analog resin. After polymerization, the coverslip glass was etched away with hydrofluoric acid. Ultrathin sections were cut using a Diatome diamond knife (Diatome, USA, Hatfield, PA) on a Leica Ultractu S ultramicrotome (Leica, Vienna, Austria). Sections were collected on copper grids and further contrasted with lead citrate, and viewed on a JSM 100 CX-II electron microscope (JEOL, USA, Inc., Peabody, MA) operated at 80 kV. Images were recorded on Kodak 4489 Electron Image film then digitized on an Epson Expression1600 Pro scanner at 900dpi for publication.
Data were expressed as the mean ± SE. Differences between two groups were measured by the Student’s two-tailed, unpaired t test, with significance set at P<0.05. When these two groups were followed over time, an ANOVA with repeated measurements followed by Fisher post hoc test was used, and significance set at P<0.05.
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 (Fig. 1A). 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 (Fig. 1B).
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 (Fig. 1C, 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) (Fig. 1C, right panels).
Then, we demonstrated the effectiveness of mitoDendra in studying the dynamics of mitochondria in primary neurons by time-lapse in vivo imaging (Fig. 1D). 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 figure 1D, the neuronal mitochondrial network underwent extensive fusion, as demonstrated by the mixing of red and green fluorescence (Fig. 1D). We also determined that repetitive imaging did not cause photo-bleaching of mitoDendra fluorescence (Fig. 1D).
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 (Fig. 2A, top panels). However, in G93A SOD1 motor neuron somas, yellow fluorescence was not apparent until 100 min after photo-activation (Fig. 2A, 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 (Fig. 2B).
In the motor axons, identified by morphological criteria, fusion events involving individual mitochondria could be visualized during time-lapse recordings without applying photo-activation (Fig. 2C, 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 (Fig. 2C).
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.
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 (Fig. 2D) 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 (Fig. 2E) and red (Fig. 2F) 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 (Fig. 3A). 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.
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 (Fig. 3B), 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 (Fig. 3C; P = 0.016 by Student’s t test; n = 18–27 axons), while there were no significant differences in the duration of the pauses (Fig. 3D). Moreover, the velocities in the retrograde direction were reduced in mutant motor neurons (Fig. 3E; 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 (Fig. 3F), the distance traveled by mobile mitochondria was shorter in G93A motor neurons (Fig. 3G; 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 (Fig. 3H; 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 (Fig. 4A) (Koo et al., 1990; Kaether et al., 2000). Kymographs of APPDendra labeled MBO demonstrated higher motility of these vesicles (Fig. 4B) 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 (Fig. 3C). Velocity of MBO transport was also unaffected (Fig. 3D), 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.
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 (Fig. 5A,B), 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 (Fig. 5B), 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 (Fig. 5C), indicating that mitochondrial morphological abnormalities were limited to motor axons.
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 (Fig. 5D). 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 (Fig. 5E, top panels) and electron microscopy (Fig. 5E, 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 (Fig. 5F).
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 (Fig. 5G, P = 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.
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 (Fig 6A). 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 (Fig. 6B), and in either anterograde or retrograde directions (not shown).
The average mitochondrial length in both proximal and distal axonal segments was also unchanged in G93A SOD1 cortical neurons at 8 div (Fig. 6C,D), 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 (Fig. 6E). The relative amount of G93A SOD1 in purified cortical neurons was higher than in motor neurons (Fig. 6F), excluding that the lack of mitochondrial abnormalities in cortical neurons was related to lower mutant protein load.
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 (Fig. 7A, P = 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 (Fig. 7A, P = 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).
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 (Fig. 7B). 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 (Fig. 7C). 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.
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 (Fig. 8A–C).
To determine whether abnormal mitochondrial distribution was associated with the observed synaptic abnormalities, we measured the colocalization of mitochondria and SV2-labeled puncta (Fig. 8D). 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 (Fig. 8E) and the puncta mitochondrial density (Fig. 8F) 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; Fig. 8G,H), 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.
We used time-lapse confocal microscopy and mitoDendra labeling to investigate mitochondrial dynamics in motor neurons from transgenic rats expressing ALS-linked G93A mutant or WT SOD1. We identified abnormalities involved in fundamental aspects of mitochondrial biology, such as fusion and transport, in mutant, but not in WT, SOD1 motor neurons. In association with these defects, mitochondria in axons had decreased size and density, and impaired bioenergetics. Moreover, abnormal mitochondrial distribution in mutant SOD1 motor neurons correlated with neuron-to-neuron synaptic dysfunction.
Mitochondrial fusion is a process that appears to be necessary for the maintenance of healthy mitochondria and to ensure neuronal viability (Chen and Chan, 2006; Chen et al., 2007). By time-lapse microscopy of individual motor neurons, we showed decreased mitochondrial fusion in cell bodies and axons of mutant SOD1 motor neurons. Defective fusion may derive from impaired transport, if mitochondria are not brought in close proximity then fusion can not be initiated. On the other hand, it is possible that defective fusion is a primary pathogenic event, leading to defective mitochondrial morphology and function, which then results in transport defects. In favor of this interpretation, ablation of Mfn2 gene in neurons has been shown to cause mitochondrial hyper-fragmentation and impaired transport (Chen et al., 2007).
The apparent transport defects affected mitochondria but not MBO, which were normally trafficked along motor neuron axons. This indicates that mutant SOD1 does not impair fast axonal transport globally, but selectively targets specific organelles. Furthermore, mitochondrial abnormalities appeared to spare dendritic processes of motor neurons as well as cortical neuron axons, suggesting that motor neuron axons are highly susceptible to mitochondrial damage induced by mutant SOD1. The selective impairment of mitochondrial transport in spinal motor neurons may relate to a preferential binding of mutant SOD1 to mitochondria of spinal cord (Liu et al., 2004; Pasinelli et al., 2004), although the molecular determinants of these interactions involved in transport defects remain to be further investigated.
Earlier studies of mitochondrial transport in mutant SOD1 models had shown defects in anterograde axonal transport of mitochondria (De Vos et al., 2007). In those studies, non-transgenic rat cortical neurons were investigated after transient transfection with exogenous human mutant SOD1, which resulted in impaired mitochondrial transport. In our study, we did not detect mitochondrial transport or morphological abnormalities in primary cortical neurons derived from the same strain of mutant SOD1 transgenic rats used for the spinal motor neuron studies. The difference between our results and those by De Vos et al. may be due to our use of a homologous model system, where expression levels of the mutant protein were driven by the same transgenic construct in all tissues. Also, the main direction of the motility defect was different, since we identified a defect predominantly in retrograde transport. Our results are consistent with the finding of Zhang et al. that an aberrant interaction exists between mutant SOD1 and components of the dynein complex that are involved in retrograde mitochondrial transport (Zhang et al., 2007). Furthermore, vesicular axonal retrograde transport defects have been described in detail in motor neurons expressing mutant SOD1 by Perlson et al. (Perlson et al., 2009). MBO axonal transport defect was also reported in both anterograde and retrograde directions in cortical neurons transiently transfected with mutant SOD1 (De Vos et al., 2007). We investigated MBO in spinal cord motor neurons and did not detect transport abnormalities. Again, these differences underline the importance of investigating homogeneous model systems.
There is evidence that argues in favor of a cause-effect relationship between mitochondrial dynamics impairment and distal motor neuron degeneration. For instance, such a relationship has been directly demonstrated in a Drosophila model, where inactivation of the cargo adaptor for fast mitochondrial anterograde transport, dMiro, resulted in mitochondrial accumulation in the cell body, NMJ degeneration, and muscle denervation and atrophy (Guo et al., 2005). Our findings seem consistent with the hypothesis that mitochondrial transport may be involved in distal motor neuron degeneration, since in mutant SOD1 motor axons mitochondrial abnormalities were prominent in the distal regions. This could be an indication that distal axonal mitochondria suffer from accelerated degeneration, as a consequence of impaired transport and fusion, possibly because of inadequate fusion with new mitochondria originated in the soma.
Impaired mitochondrial fusion and transport, and abnormal morphology correlated with reduced mitochondrial ΔΨ in mutant SOD1 motor neurons. The finding that only mitochondria moving in an anterograde direction had a reduction of ΔΨ may suggest that mitochondria moving towards the distal portions of the axon were damaged by mutant SOD1, and incapable of providing for the bioenergetic needs of the axonal terminals and to reactivate aging mitochondria by fusing with them. These findings agree with the observations that mutant SOD1 axons start degenerating at their terminals, where damaged mitochondria accumulate early on in the course of degeneration (Vande Velde et al., 2004; Gould et al., 2006).
Mitochondrial bioenergetic impairment has been extensively demonstrated in the mutant SOD1 mouse (for a review see (Magrane and Manfredi, 2009)), but the causal relationship between impaired bioenergetics and defective transport needs to be further clarified. While it is possible that the two defects originate independently and proceed in parallel, it might also be that transport defects contribute to the bioenergetic problem. It could be hypothesized that mitochondria are not effectively regenerated and fail to maintain sufficient metabolic activity, and eventually succumb when challenged with heavy workloads, such as buffering of intracellular Ca2+ spikes. Dysfunctional mitochondria that are not efficiently recycled by fusion with healthy mitochondria may become fragmented and targeted for mitophagy (Twig et al., 2008).
The molecular determinants of impaired mitochondrial dynamics in mutant SOD1 motor axons have not yet been characterized. We observed that mutant SOD1 caused a predominant effect on retrograde transport of mitochondria, with reduced motility, increased frequency of pauses, decreased velocity and decreased distance traveled. The observation that some of these abnormalities were present predominantly in either proximal or distal regions of the axons suggests that different players may be involved in regulating transport of mitochondria in these regions. Furthermore, the balance between competing anterograde and retrograde forces driving organelle transport (Hendricks et al., 2010; Schuster et al., 2011; Ally et al., 2009) could be altered, if mutant SOD1 interferes preferentially with the molecular machinery for anterograde or retrograde transport. Normal MBO transport seems to exclude abnormalities in microtubule tracks, which would likely impair the transport of all cargoes and not mitochondria specifically. In addition to a direct interaction with the transport machinery, it is possible that SOD1 interferes with the complex signaling mechanisms regulating mitochondrial motility. For example, inactivation of the cargo adaptor Miro1 by increasing local Ca2+ concentration has been recently identified (Liu and Hajnoczky, 2009), suggesting that impaired mitochondrial Ca2+ handling in mutant SOD1 motor neurons (Kawamata and Manfredi, 2010) could contribute to transport defects. Furthermore, mutant SOD1 may affect mitochondrial motility by impairing the regulation of molecular motors by cytosolic kinases (Bosco et al., 2010; De Vos et al., 2000; Morfini et al., 2006).
What are the consequences of mitochondrial mislocalization and dysfunction in motor neuron axons? Published reports suggest that mitochondrial Ca2+ uptake is impaired in brain, spinal cord and motor nerve terminals in G93A SOD1 mice (Vila et al., 2003; Damiano et al., 2006). Furthermore, in pre-symptomatic mutant mice, an altered response to Ca2+ loads in mitochondria within motor neurons involving altered mitochondrial membrane potential has been described (Nguyen et al., 2009). Based on these findings and on our observations of mitochondrial dynamics abnormalities, it is possible that mitochondrial Ca2+ handling is defective at the NMJ of mutant SOD1 mice, resulting in impaired local Ca2+ homeostasis. Damaged mitochondria may also be incapable of providing adequate ATP supplies for synaptic transmission, and may become the source of toxic free radicals. Although the motor neuron-to-motor neuron synaptic changes observed in vitro have unknown pathological significance for ALS, the correlation between abnormal mitochondrial distribution at pre-synaptic sites and reduction of size, number, and function of synapses supports the view that mitochondrial dynamics defects may contribute to synaptic abnormalities.
Taken together, our observations suggest that perturbation of axonal mitochondrial dynamics may contribute to motor neuron degeneration in familial ALS with SOD1 mutations. Future studies will have to clarify the molecular mechanisms underlying mitochondrial dynamics impairment in mutant SOD1 motor neurons. They will also need to determine if impaired mitochondrial dynamics is a common factor in other motor neuron diseases, including other familial forms and sporadic ALS.
We thank Dr. Konstantin A. Lukyanov (Russian Academy of Sciences, Moscow, Russia) for Dendra cDNA, and Leona Cohen-Gould (Cornell University, New York, NY, USA) for preparation and imaging of samples for transmission electron microscopy. We also wish to thank Dr. Geri E. Kreitzer (Cornell University, New York, NY, USA) and Dr. Roy C. Smith (Tufts University, Boston, MA) for discussion and critical reading of the manuscript. This work was supported by grants from the Muscular Dystrophy Association (to J.M., S.P., G.M.,), the Robert Packard Center for ALS Research “The New York Community Trust” (to G.M.), the National Institutes of Health (NS051419, NS062055 to G.M.; NS042269, NS064191, NS38370, NS070276, and NS072182 to S.P.; NS36761 to A.G.E), the Department of Defense (W81XWH-08-1-0522, W81XWH-08-1-0465, W81XWH-09-1-0245 to S.P.), and Project A.L.S (to S.P.).
Conflict of Interest: The authors declare no conflict of interest.