In the case of axonal transport, it may not be about who wins the race, but what’s carried to the finish line. Retrograde axonal transport slows down in motor neurons in animal models of amyotrophic lateral sclerosis (ALS), depriving the cell body of neurotrophic factors. But the real problem, according to a paper in the August 5 Journal of Neuroscience, is that when the dynein-powered retrograde transporters finally get to the cell body, it is not those feel-good neurotrophins they deliver, but stress signals that cause apoptosis. First author Eran Perlson and principal investigator Erika Holzbaur, both of the University of Pennsylvania in Philadelphia, and colleagues suggest that the cargo itself is key to the rapid decline of ALS. In other ALS news, two recent papers in the Journal of Biological Chemistry may help to explain the mechanism of protein aggregation that underlies some forms of the disease.
Mice expressing mutant human superoxide dismutase 1 (SOD1), an enzyme sometimes mutated in people with familial ALS, are a common model for the condition. In these animals, retrograde transport is inhibited, and animals die well before reaching a year of age. Yet there are other animal models with sluggish retrograde transport, and they suffer milder neurodegeneration than mSOD1 animals. The Loa mouse, with a point mutation in dynein, and a mouse that overexpresses the dynein inhibitor dynamitin, both exhibit transport delays without such severe disease (see ARF related news story on Hafezparast et al., 2003 and ARF related news story on LaMonte et al., 2002). Perlson and colleagues sought to determine what makes disease in mSOD1 animals so much worse.
First, the researchers analyzed transport in mSOD1-G93A animals using sciatic nerve ligation assays. Retrograde travel of dynein subunits was reduced by approximately 30 percent compared to that in mice expressing wild-type SOD1. In previous studies, retrograde transport in Loa and dynamitin-overexpressing mice was reduced by a similar amount. Perlson and colleagues also used live imaging to monitor vesicle movement in embryonic rat motor neuron cultures. In cells expressing mSOD1-G85R, retrograde transport slowed to 0.41 microns per second, compared to 0.76 microns per second in cells expressing wild-type SOD1. Transport of specific neurotrophic cargoes, such as the nerve growth factor NRG, was also inhibited. The Loa and dynamitin mice showed similar deficiencies in growth factor trafficking.
The scientists reasoned that if the transport speed is not the difference, perhaps cargo is the issue. To assess what dynein was carrying, Perlson and colleagues performed dynein pulldowns on axoplasm-enriched fractions from 85-day-old mSOD1 and control non-transgenic mice. They used an antibody microarray to identify protein levels that differed between the two. Of the 102 proteins that linked less often with dynein in mSOD1 animals, 46 percent were associated with promoting cell survival. Conversely, among the 52 proteins that associated more frequently with dynein in the mSOD1 mice, 44 percent were associated with cellular stress. “Right away, the answer fell out of the analysis,” Holzbaur said. “Axonal transport is clearly going from a survival signaling pattern to a stress signaling pattern.”
Using directed pulldown assays and immunoblotting to confirm the results from the microarrays, the researchers determined that pro-survival molecules such as P-Trk, P-Erk1/2, and P-Erk5 were reduced by approximately 60 percent in sciatic nerve extracts from mSOD1 animals, compared to the levels in non-transgenic mice. Activated caspase-8, P-JNK, and p75-NTR cleavage products were increased in the mSOD1 dynein pulldowns, suggesting the motor protein toted these cell stress signals to the cell body. Neurodegeneration is likely a result of all of these changes, Perlson said: “There is not one magic pathway.”
If stress signals moving up the axon are the cause of cell damage, then inhibiting those signals should save the cell. To test this idea, the researchers used a compartmentalized cell culture system, with motor neurons growing in sections adjacent to glial cells. Axons could extend across the barriers. After six days in culture, half as many mSOD1-expressing cells survived as those expressing wild-type SOD1 or no transgene. The researchers then added specific inhibitors to JNK, caspase-8, and p75-NTR. Under these conditions, survival of mSOD1 cells rose to 86 percent of control levels. Overexpression of dynamitin, to slow transport, was not as effective protection as the inhibitors, suggesting that when transport was slowed, some stress signals still made it to the cell body. This supports the hypothesis that cell stress signaling, not slowed transport, is the real killer. Other experiments indicated that the stressful communiqués come from glia, as mSOD1 neurons cultured alone did not experience the uptick in P-Trk levels associated with cell stress and death. This fits with the popular theory that motor neuron death in ALS is non-cell-autonomous (reviewed in Boillée et al., 2006).
The work suggests that fixing the transport problem alone would not be a valid ALS treatment, nor would providing motor neurons with additional neurotrophic factors. A therapeutic would also have to block the stress signaling, Perlson suggested.
“It is a whole new hypothesis as to what is going on,” said Julie Andersen of the Buck Institute for Research in Aging in Novato, California. Yet the research says little about how axonal dynein comes by the additional stress factors. “To me, that is kind of the million-dollar question,” she said. The mechanism is key, agreed Scott Brady of the University of Illinois in Chicago. “What remains unclear is how mutant SOD1 affects the interaction of dynein with such a wide variety of transported cargoes,” he wrote in an e-mail to ARF. “Further, we need to understand what mutant proteins like SOD1 do to affect motor function.” Neither Andersen nor Brady was involved in the current study.
What mSOD1 does is a question many are addressing with biochemistry and structural biology studies, but the puzzle is compounded by the fact that more than 100 different SOD1 mutations can cause ALS. “We’re all looking for the one gain of function, the one thing that all the mutants have in common,” said Jeffrey Agar of Brandeis University in Waltham, Massachusetts. Agar and colleagues published a paper in the Journal of Biological Chemistry July 27 suggesting that a freely flapping electrostatic loop could be that common factor. The loosened structure could make the protein more likely to form intermolecular bonds leading to dangerous aggregates. Agar, first author Kathleen Molnar of the ExSAR Corporation in Monmouth Junction, New Jersey, and colleagues analyzed 13 different SOD1 mutants. “We were not expecting this when we started, believe me, but in all of them that electrostatic loop is floppier or more dynamic,” Agar said.
Another paper in the Journal of Biological Chemistry August 3 confirms the importance of SOD1’s metal cofactors in maintaining the protein’s stability. Principal investigator Lawrence Hayward of the University of Massachusetts Medical School in Worcester and first author Ashutosh Tiwari, who recently moved from Hayward’s lab to start his own at the Michigan Technological University in Houghton, led this study and were also coauthors on Agar’s July 27 paper. By analyzing the exposure of hydrophobic residues in different forms of the enzyme, the authors found that both zinc and copper ions were necessary to fully stabilize SOD1, making it less aggregation-prone.—Amber Dance
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- Dynamitin in Motor Neurons: Dynamite for ALS Research?
- Role of the Motor in Motor Neuron Diseases
- Huntingtin—Putting the Boot on Axonal Transport
- ALS—Is It the Neurons or the Glia?
- Frustrated ALS Enzyme: SOD1 Sacrifices Structural Stability for Function
- Following SOD1 Biochemistry in ALS from Start to Finish
- Runaway Train: Mutations in Dynactin’s Brake Cause Rare Syndrome
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