Oxidative stress inhibits axonal transport: implications for neurodegenerative diseases
© Fang et al.; licensee BioMed Central Ltd. 2012
Received: 24 February 2012
Accepted: 18 June 2012
Published: 18 June 2012
Reactive oxygen species (ROS) released by microglia and other inflammatory cells can cause axonal degeneration. A reduction in axonal transport has also been implicated as a cause of axonal dystrophies and neurodegeneration, but there is a paucity of experimental data concerning the effects of ROS on axonal transport. We used live cell imaging to examine the effects of hydrogen peroxide on the axonal transport of mitochondria and Golgi-derived vesicles in cultured rat hippocampal neurons.
Hydrogen peroxide rapidly inhibited axonal transport, hours before any detectable changes in mitochondrial morphology or signs of axonal degeneration. Mitochondrial transport was affected earlier and was more severely inhibited than the transport of Golgi-derived vesicles. Anterograde vesicle transport was more susceptible to peroxide inhibition than retrograde transport. Axonal transport partially recovered following removal of hydrogen peroxide and local application of hydrogen peroxide inhibited transport, suggesting that the effects were not simply a result of nerve cell death. Sodium azide, an ATP synthesis blocker, had similar effects on axonal transport, suggesting that ATP depletion may contribute to the transport inhibition due to hydrogen peroxide.
These results indicate that inhibition of axonal transport is an early consequence of exposure to ROS and may contribute to subsequent axonal degeneration.
KeywordsHydrogen peroxide Oxidative stress Axonal transport Mitochondria Golgi-derived vesicles Neurodegeneration
Axonal transport is critical for maintaining axonal integrity. Anterograde axonal transport supplies the axon with proteins synthesized in the cell body and retrograde transport delivers endosomal signaling organelles from the terminal to the cell body . Interrupting axonal transport leads to degeneration of the distal axon, much like the Wallerian degeneration that occurs after axonal transection . Axonal transport is mediated by kinesins and dyneins, motor proteins that use ATP hydrolysis to power their translocation along microtubules [3, 4]. Mutations that disrupt kinesin- or dynein-mediated transport lead to human diseases characterized by axonal dysfunction , such as some forms of Charcot-Marie-Tooth disease  and hereditary spastic paraplegia . Alterations in axonal transport have also been implicated in neurodegenerative diseases, including Alzheimer’s disease , amyotrophic lateral sclerosis , Parkinson’s disease , and Huntington’s disease .
Oxidative stress, one of the major characteristics of neuro-inflammation, which results from the unregulated production of reactive oxygen species (ROS), has also been implicated in many neurodegenerative diseases [12–15]. Activated microglia, the resident innate immune cells in the brain, release ROS and reactive nitrogen species (RNS), including hydrogen peroxide, nitric oxide, peroxynitrite and superoxide . Microglial activation occurs in many neurodegenerative diseases and in the neuroinflammatory diseases, such as multiple sclerosis [17, 18]. Emerging evidence suggests that increased levels of ROS play an important role in triggering axonal degeneration [12–14]. Given the importance of axonal transport for the preservation of axonal integrity, surprisingly little is known about how oxidative stress affects axonal transport and whether this contributes to the damaging effects of elevated levels of ROS.
New methods based on live cell imaging make it possible to evaluate the efficiency of axonal transport with an increased level of precision . We used this approach to investigate the effects of one ROS, hydrogen peroxide, a byproduct of mitochondrial oxidative metabolism that is elevated in many neurodegenerative diseases .
We found that hydrogen peroxide rapidly inhibited axonal transport of both mitochondria and Golgi-derived vesicles, providing the first direct evidence for the ability of ROS to inhibit axonal transport.
Transport characteristics of mitochondria and Golgi-derived vesicles
Hydrogen peroxide-induced inhibition of axonal transport occurs hours before the first signs of axonal degeneration
Figure 3C and 3D summarize the effects of hydrogen peroxide on vesicle transport, based on an analysis of 20 cells. Hydrogen peroxide caused a significant reduction in the velocity of both anterograde and retrograde transport (which reached statistically significant levels after about 35 minutes of exposure) and in the number of anterograde transport events (which reached statistical significance after 50 minutes). There was no reduction in the number of retrograde events. This “signature” of hydrogen peroxide damage—a reduction in the velocity of both anterograde and retrograde transport but a selective reduction in the number of anterograde events—was apparent in the recordings from nearly every cell. Since anterograde transport was preferentially inhibited, the anterograde over retrograde ratio fell from a mean of 1.7:1 before exposure to 0.7:1 after exposure for 1 h.
A comparison of Figure 3D and 4B clearly indicates that hydrogen peroxide has very different effects on the transport of Golgi-derived vesicles compared with mitochondria. Mitochondrial transport was much more sensitive to the effects of hydrogen peroxide and anterograde and retrograde mitochondrial transport were both affected similarly. Inhibition of vesicle transport required longer exposures to hydrogen peroxide and anterograde vesicle transport was more severely affected.
As expected, the effects of hydrogen peroxide on axonal transport were dose-dependent. At higher concentrations (250 μM hydrogen peroxide), the inhibition of transport occurred more rapidly and the number of vesicles moving in both the anterograde and retrograde directions was significantly reduced. As at lower concentrations of hydrogen peroxide, anterograde vesicle transport was more severely affected. Anterograde transport was inhibited by 70% after about 35 minutes, while a comparable inhibition of retrograde transport required exposure for 70 minutes. Lower concentrations of hydrogen peroxide had less dramatic effects. Exposure to 20 μM and 50 μM hydrogen peroxide inhibited anterograde vesicle transport by 20% and more than 30% respectively after 1 hour and had no effects on retrograde transport in both concentrations. For any given concentration of hydrogen peroxide, mitochondrial transport was more sensitive to hydrogen peroxide than was the transport of Golgi-derived vesicles.
Local exposure to hydrogen peroxide inhibits axonal transport
Axonal transport recovers partially after hydrogen peroxide exposure
In contrast to mitochondrial transport, anterograde transport of Golgi-derived vesicles, which dropped by about 50% after treatment with 100 μM hydrogen peroxide, was fully restored after recovery for 1 hour (Figure 6B). Moreover, the velocities of anterograde and retrograde vesicle transport also returned to control levels after 1 hour recovery (Figure 6C). The recovery of vesicle transport was also dose-dependent. Following exposure to 250 μM hydrogen peroxide, anterograde transport of Golgi-derived transport was reduced to less than 20% of controls and retrograde transport was also reduced (to about 30% of controls). Transport of Golgi-derived vesicles recovered partially after return to control media for 1 hour, but failed to reach control levels. Retrograde transport of Golgi-derived vesicles was less severely inhibited than anterograde transport and recovered more completely (data not shown). Taken together, these data suggest that axonal transport recovers from mild but not from severe hydrogen peroxide–induced inhibition. For any given concentration of hydrogen peroxide, mitochondrial transport is more sensitive to hydrogen peroxide than is the transport of Golgi-derived vesicles and is less likely to recover.
Hydrogen peroxide-induced axonal degeneration precedes neuronal cell death
Changes in mitochondrial morphology have been reported to correlate with axonal damage and are thought to be one of the earliest signs of axonal degeneration, preceding changes in axonal morphology . We wondered whether hydrogen peroxide caused changes in mitochondrial morphology prior to its effects on axonal transport. Mitochondrial morphology was evaluated by computing the shape factor, an indicator of how similar an object is to a perfect sphere, which has a shape factor of 1.0. Even after 1 hour of exposure to hydrogen peroxide, when mitochondrial transport was reduced by 90%, there was no change in mitochondrial shape (shape factor: control 0.78 ± 0.19; 100 μM hydrogen peroxide for 1 hour 0.78 ± 0.22; n = 173 and 147 mitochondria, respectively). These data show that axonal transport inhibition precedes changes in mitochondrial morphology.
Given these results, we wondered whether dendritic transport of mitochondria and Golgi-derived vesicles was less sensitive to hydrogen peroxide than axonal transport. As shown in Figure 8B, the transport of Golgi-derived vesicles was profoundly inhibited in both the dendrites and the axon. These results suggest that the initial effects of hydrogen peroxide exposure are similar in axons and dendrites, but the long-term consequences of this exposure preferentially affect the axon, ultimately leading to its degeneration.
ATP depletion reversibly inhibits axonal transport and does not induce axon degeneration
Axonal transport is ATP-dependent  and in vitro assays show that the motors that mediate axonal transport hydrolyze 1 molecule of ATP for every step they take along the microtubule . Since hydrogen peroxide damages mitochondria and causes depletion of ATP , we set out to determine whether ATP depletion contributes to the inhibition of axonal transport and the axonal degeneration that occur following hydrogen peroxide exposure. To address this issue, we treated hippocampal cultured neurons with sodium azide (NaN3), a specific inhibitor of cytochrome C oxidase, complex IV of the mitochondrial respiratory chain.
We also examined whether the axonal transport returned to normal levels following ATP depletion. Following exposure to azide for 1 h, the transport of both mitochondria and Golgi-derived vesicles (velocity as well as event numbers) recovered completely (Figure 9A). This is quite different from the effects of hydrogen peroxide treatment, which permits only a partial recovery of mitochondrial axonal transport. Moreover, one hour of sodium azide treatment did not induce axonal degeneration. Axons remained intact even at 24 hours after treatment, as shown by beta3-tubulin staining (Figure 9B). Taken together, these data suggest that ATP depletion may contribute to some of the effects of hydrogen peroxide on axonal transport, but the latter agent leads to irreversible changes in the machinery underlying axonal transport that are not due simply to ATP depletion.
This report is the first direct demonstration of the ability of hydrogen peroxide to inhibit axonal transport. Using live-cell imaging of cultured hippocampal neurons, we found that hydrogen peroxide profoundly inhibited the axonal transport of both mitochondria and Golgi-derived vesicles. Surprisingly, mitochondrial transport was affected earlier and was more severely inhibited than vesicle transport.
The paradigm we used—live-cell imaging of cultured neurons--allowed us to rigorously characterize the effects of hydrogen peroxide on axonal transport, but it required that we use comparatively high concentrations, so that the effects on transport could be observed within an hour or so. These concentrations of hydrogen peroxide eventually lead to axonal degeneration and ultimately nerve cell death, but several lines of evidence suggest that the hydrogen peroxide-induced inhibition of axonal transport we observed is not simply the response of a dying cell. First, the inhibition of transport occurred relatively early, even before a change in mitochondrial morphology, which is thought to be among the earliest signs of ROS-induced axonal damage . Second, the effects were seen after local exposure of axons to hydrogen peroxide in microfluidic chambers in which the nerve cell bodies are not exposed to hydrogen peroxide. Third, after removal of hydrogen peroxide, vesicle transport returned to near-normal levels. If the inhibition of transport was a secondary consequence of axonal degeneration, one would expect it to worsen over time, not to improve. Fourth, different components of axonal transport were differentially affected. It seems unlikely that transport failure secondary to axonal degeneration would inhibit anterograde vesicular transport but spare retrograde transport. Finally, similar but milder effects on axonal transport were observed during a 1-hour exposure to lower concentrations of peroxide. Under these conditions, the great majority of axons show no evidence of morphological damage one day after treatment.
How does hydrogen peroxide exposure lead to inhibition of transport?
Hydrogen peroxide, like other ROS, disrupts many cellular processes, including mitochondrial ATP production and regulation of calcium homeostasis , ion channel permeability , and redox signaling . At present, we do not know the pathways that lead to inhibition of axonal transport. Some of the effects of hydrogen peroxide on axonal transport were quite similar to those produced by sodium azide, an inhibitor of ATP production . Following both treatments, mitochondrial transport was inhibited first, then anterograde vesicle transport, and then retrograde vesicle transport. Thus it is reasonable to attribute some of the effects of hydrogen peroxide exposure to ATP depletion. Several of our findings, however, suggest that there is more involved than simply a reduction in the ATP levels available to molecular motors. Since kinesins and dyneins both have similar requirements for ATP, the differential effects on anterograde versus retrograde transport are more likely to involve the many signaling pathways activated by increased levels of ROS than simply a reduction in ATP. For example, stress-activated kinases  can phosphorylate kinesins and inhibit their translocation, which could lead to a selective inhibition of anterograde transport.
Why is mitochondrial transport preferentially inhibited by hydrogen peroxide and sodium azide? One possibility relates to the molecular adaptors that link mitochondria to kinesins and enable cytoplasmic calcium levels to regulate mitochondrial transport. Miro, an EF-hand containing protein in the outer mitochondrial membrane, binds to Milton, a cytoplasmic adaptor protein that in turn binds to the heavy chain of Kinesin-1 [33, 34]. When cytoplasmic calcium levels increase, calcium binds to the EF hands on Miro, permitting Miro to bind to the motor domain of kinesin-1 and stopping mitochondrial transport [35, 36]. Both hydrogen peroxide treatment and ATP depletion disrupt mitochondrial calcium buffering and elevate cytoplasmic calcium levels [29, 30], which would be expected to activate Miro. In contrast, Golgi-derived vesicles are linked to Kinesin-1 via interactions that are not calcium-dependent.
We focused our work on the effects of hydrogen peroxide because it is elevated in some models of neuroinflammatory and neurodegenerative diseases [37–39] and because it is easy to manipulate in vitro. However, based on similarities in their mechanisms of action, it would not be surprising if other ROS and RNS had similar effects on axonal transport. Stagi et al  reported that exposure to nitric oxide inhibited axonal transport of synaptic vesicle proteins in cultured hippocampal neurons, although the fluorescence correlation spectroscopy method they used did not allow characterization of the effects at the level of individual vesicle movements. Although the molecular mechanism of hydrogen peroxide-induced axonal transport inhibition is not clear, activation of stress-activated kinases can phosphroylate kinesins, inhibiting axonal transport [40, 41]. Activation of local apoptotic pathways could also be involved .
Could ROS-induced inhibition of transport contribute to axonal disease?
Our results indicate that hydrogen peroxide exposure preferentially inhibits mitochondrial trafficking and the anterograde transport of Golgi-derived vesicles, including those that carry proteins essential for maintaining axonal integrity . Retrograde transport, which delivers trophic factors required for cell survival, is less affected. Over the long term, if axons were exposed to local increases in ROS species, this pattern of transport inhibition could result in the dying-back axonal degeneration that characterizes several neurodegenerative diseases, including the progressive phase of some neuroinflammatory diseases. Our results also show that axons are more susceptible to ROS damage than nerve cell bodies and dendrites, so it is possible that more global changes in inflammation, which are hypothesized to play a role in several neurodegenerative diseases [12–15], could preferentially affect axons.
While there may be some situations in vivo where axons are exposed to high concentrations of hydrogen peroxide or other ROS , in most diseases that have an inflammatory component axons are likely to be chronically exposed to modestly increased levels of ROS. It remains to be seen if the effects we observed following acute exposure to hydrogen peroxide are mimicked during chronic exposure to lower levels of this agent or other ROS. To the extent it was possible to examine the dose–response characteristics in our model, we observed similar effects at lower concentrations of hydrogen peroxide, but they were less severe and occurred over a slower time course. If ROS-mediated inhibition of axonal transport contributes to neural disease in vivo, the cell culture model we describe here for analyzing axonal transport could serve to elucidate the molecular details of how ROS disrupt axonal transport and suggest potential neuroprotective therapies.
In this study, we show that exposing cultured hippocampal neurons to hydrogen peroxide, one of the common ROS elevated during inflammation, causes an inhibition of axonal transport several hours before any signs of axonal degeneration occur. The transport of different organelles was differentially affected—mitochondrial transport was affected earlier and more severely than the transport of Golgi-derived vesicles; anterograde transport of Golgi-derived vesicles was inhibited more severely than retrograde transport. Local exposure of axons to hydrogen peroxide was sufficient to inhibit axonal transport and induce axonal degeneration. These results raise the possibility that ROS and RNS-induced inhibition of axonal transport in apparently healthy axons could contribute to the dying-back axonal degeneration that occurs in some neurodegenerative diseases.
Materials and methods
Primary neuronal culture and transfection
Primary hippocampal cultures were prepared from E18 embryonic rats of either sex as described previously [43, 44] Cells were plated at 75 cells/mm2 on poly-L-lysine-treated coverslips and maintained in MEM supplemented with N2. For electroporation, 0.5-1 μg of DNA was introduced into the neurons through electroporation at DIV0 before seeding and then the neurons were seeded at 500 cells/mm2. Neurons were imaged at DIV7-10. For transfection, 1–2 μg of DNA was introduced to the neurons at 7–8 days in vitro (DIV) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Neurons were imaged 16–20 hours after transfection.
Live-dead assay was performed following company protocol (Invitrogen, Carlsbad, CA). Briefly, Calcein AM and EthD-1 were added directly to neuronal culture medium (final concentration 2 μM of calcein AM and 4 μm of EthD-1). Cells were returned to the incubator for 20 minutes. Following incubation, cells were gently rinsed with warm PBS and changed into fresh neuronal culture medium and immediately brought to the microscope. Cells were maintained at 37 degrees for live imaging.
For immunostaining, DIV7-10 neurons were fixed in 4% paraformaldehyde, 4% sucrose for 15 minutes, permeabilized in 0.25% Triton X-100 for 5 minutes prior to primary antibodies (Tuj-1, Sigma; Map2b, Sigma; Tau-1, Sigma) 4 degrees overnight followed by secondary antibodies (488 anti Mouse, Cy3 anti Rabbit, Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 45 minutes. The coverslips were mounted in elvanol .
All constructs were expressed from the plasmid vector containing a beta-actin promoter/enhancer . SS-mCherry was engineered to have secretory sequence of neuropeptide Y (NPY)  fused to the N terminus of red fluorescent protein mCherry. Mito-GFP was constructed by insertion of a mitochondria targeting sequence from subunit VIII of human cytochrome c oxidase to the N terminus of eGFP. All constructs were verified by sequencing.
Imaging and analysis
Before acquiring movies, coverslips were semi-sealed into a heated chamber (Warner instruments, Hamden, CT) containing hibernate A medium (Brainbits LLC, Springfield, IL) kept at 37 degree. Images were captured with a spinning disk confocal microscope setup custom built by Solamere Technology Group (Salt Lake City, Utah). Laser excitation wave length for different fluorophores was 488 nm for GFP and 568 nm for mCherry. Mitochondrial transport was acquired using a 40X 1.3 N.A oil objective. Golgi-derived vesicles transport was acquired using a 60X 1.45 N.A oil objective. Both objective and the imaging stage were heated to 37 degrees.
To perform analysis of organelle transport, we used kymograph function in Metamorph. For each movie frame, the brightest pixel within a 2 μm corridor along the axis of an axon (or dendrite) is displayed at the corresponding location of a kymograph. The fluorescence patterns for all frames are then displayed adjacent to one another. This produced a graph on which the x-axis represents time and the y-axis represents distance along the process. The diagonal lines on each kymograph indicated moving organelles and were traced on each kymograph. All organelles that moved more than 5 μm were included in the data. Event numbers were computed by the total amount of transport numbers in the stretch of axon of interest and then were normalized to every 100 μm. Statistical significance of differences between groups was determined by performing student t-test using statistical software Prism®.
Mitochondrial morphology from the same stretch of axon was examined before hydrogen peroxide treatment and at 30 min and 60 min after treatment. Regions with no or few overlapping mitochondria were picked. Mitochondrial morphology was measured as shape factor (SF) using Metamorph. SF = 4π A/P2 (P = perimeter and A = area). SF gives a value from 0 to 1 representing how closely the object represents a circle: a value near 0 indicates a flattened object, whereas a value of 1 indicates a perfect circle.
Axonal degeneration score
Axon morphology was judged by the beta3-tubulin staining of neurons. Intact axons, characterized by their thin and uniform diameter, were determined as score 0 in our axonal degeneration system. When some axonal swelling was obvious but the majority of the axons were still intact, we considered that score 1. When almost half of the axons were swelling but there was no or minimal axonal fragmentation, we considered that score 2. Score 3 indicated most of the axons in the field were swelling and some were fragmented. Finally, if all the axons were fragmented, that was considered score 4. Beta3-tubulin staining pictures of cells before and at various time points after treatment (or control) were judged by experimenter blind to the treatment conditions based on the scoring system described above. Each condition was repeated 3 times.
This research was supported by the National Multiple Sclerosis Society (MS Center Grant CA 1055-A-3), the NIH (R01NS057433), the Laura Fund for Innovation in Multiple Sclerosis and by department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development (Merit Review to DB). The contents do not represent the views of the Department of Veterans Affairs or the US government. Cheng Fang is supported by a postdoctoral fellowship from the National Multiple Sclerosis Society. We are particularly grateful to Julie Luis Harp and Barbara Smoody for their expert technical assistance, to Dr. Michael Forte, Dr. Greta Glover and Dr. Bethe Scalettar for their comments on the manuscript and to Dr. Stefanie Kaech, Dr. Helena Decker, Brian Jenkins, Dr. Marvin Bentley and Dr. Chun-fang Huang for helpful advice throughout the course of this work. The live-cell imaging was conducted at the Advanced Light Microscopy Core @ The Jungers Center, which is supported in part by NIH P30-NS06180 (S. Aicher, P.I.).
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