6 January 2012. People with amyotrophic lateral sclerosis struggle to move, and it appears some of their organelles have the same problem. Mitochondria normally sashay up and down neuronal axons, arriving fresh at the synapse and smartly returning to the cell body to be recycled. But in ALS, these organelles flounder in their travels as well as in their ability to provide the energy that neurons crave, according to new research. In the January 4 Journal of Neuroscience, researchers from the Weill Medical College of Cornell University in New York City report that in a neuron model for ALS, mitochondria exhibit a variety of defects, including lackadaisical fusion and a lollygagging passage from axon tips to the cell body.
Mitochondrial defects are associated with Alzheimer’s, Parkinson’s (see ARF related news story), and Huntington’s diseases (see ARF related news story). “This study adds ALS as one of the neurodegenerative diseases where mitochondrial dynamics is clearly affected and most likely playing a role in neurodegeneration,” said study senior author Giovanni Manfredi, who led the work with first author Jordi Magrané. Their and others’ work previously indicated a role for mitochondria (reviewed in Magrané and Manfredi, 2009) and axonal transport (De Vos et al., 2007; see ARF related news story and ARF news story) in degeneration of motor neurons in ALS, but “this is clearly the best and most comprehensive study on this topic as yet,” commented Flint Beal, who is also at Weill Medical College but was not involved in the publication.
To label mitochondria and follow their comings and goings, Magrané hooked the mitochondrial-targeting pre-sequence from cytochrome c oxidase to a photo-switchable fluorescent protein called Dendra. Dendra starts out green, but when photoactivated by blue-green light converts irreversibly to red. Magrané transfected this mitoDendra into primary embryonic motor neurons. The cells came from rats expressing human SOD1 with the G93A mutation that causes an inherited form of ALS. As controls, Magrané used rats expressing wild-type human SOD1 as well as non-transgenic animals.
To examine mitochondrial fusion, the researchers photo-converted some of the mitoDendra in each cell soma to red. Then, as green- and red-labeled mitochondria mixed, yellow mitochondria appeared, signifying fusion of organelles. In motor neurons from non-transgenic rats, it took approximately 40 minutes for yellow signals to appear. This process took more than twice as long in mSOD1 cells. Similarly, in the axons of the mSOD1 neurons, the scientists observed that mitochondria—specifically those moving in the retrograde direction—fused with each other approximately half as often as they did in control axons.
The researchers then examined mitochondrial transport along axons. In control neurons, the organelles traveled in the anterograde direction at approximately 0.2 microns per second and retrogradely at about 0.3 microns per second. While anterograde transport proceeded at normal velocity in mSOD1 cells, mitochondria coming back up in the retrograde direction traveled more slowly and paused more often than in control axons. They averaged only 0.2 microns per second. Since retrograde-moving mitochondria are destined to be recycled into fresh new organelles in the cell body, this leisurely pace could impact the production of reinvigorated mitochondria, Manfredi suggested.
Altered transport and fusion could alter the size of mitochondria, so Magrané measured the length of the organelles in axons as the cells aged. Mitochondria were shorter in the mSOD1 neurons, and the defect appeared to start at the axon tips and work toward the cell body. At five days in culture, control cells possessed mitochondria of approximately three microns in length in both proximal and distal areas. In the mSOD1 axons, the proximal mitochondria remained normal size, but the distal ones were only two microns long. After 10 days in culture, proximal mitochondria stretched to four microns in non-transgenic neurons, but only 3.3 microns in mSOD1 neuronal cultures.
This distal-first sequence of events in culture mirrors the progression of ALS pathology, in which atrophy appears to begin at the neuromuscular junction and progress to the cell body in a “dying back” of the axon (Fischer et al., 2004), noted Xiongwei Zhu and Xinglong Wang, of Case Western Reserve University in Cleveland, Ohio, in an e-mail to ARF. The data “support a critical role of mitochondria dysfunction in the dying back mechanism of the SOD1-familial ALS model,” wrote Zhu and Wang, who were not involved in the current study.
Manfredi and Magrané wondered if all retrograde transport would be affected, or only that of mitochondria. To find out, they used Dendra to label membrane-bound organelles (MBOs), vesicles that also shuttle up and down axons. MBO transit was unaffected by mSOD1, suggesting the axon’s rails and engines must be intact. Mitochondrial-trafficking deficiency also only appeared in motor neurons, Magrané found. When he examined mitochondrial dynamics in primary cortical neurons, transport was normal in the mSOD1-expressing cells.
The researchers do not yet know how mSOD1 alters mitochondrial transport. It might interrupt the organelles’ interactions with microtubules or molecular motors, Manfredi speculated (Ström et al., 2008). While mSOD1 is found all over the cell, he and Magrané have previously shown that when the mutant protein is targeted solely to mitochondria, it recapitulates many features of ALS (see ARF related news story on Igoudjil et al., 2011; Magrané et al., 2009). Therefore, it is possible that some intrinsic deficiency in mSOD1-toting mitochondria affects their morphology and transport.
“I think this work will solidify the concept that impairment of mitochondrial dynamics in axons can be a significant contributing factor to ALS etiology,” said Haining Zhu of the University of Kentucky in Lexington, who was not involved in the study. And mutant SOD1 is likely not the only way to muck up mitochondrial transport. Other rodent models for ALS, based on mutations in genes such as TDP-43, also exhibit abnormal mitochondrial morphology (see ARF related news story on Xu et al., 2010). Those genetic mutations, however, account for only a small percentage of human ALS. Most cases are sporadic, and mitochondrial transport might turn out to be amiss in those motor neurons, too, Beal suggested. “I think it is feasible that it is going to be a generalized problem,” he said.
Moreover, mitochondrial defects are emerging as a common thread among neurodegenerative disease. “Neurons are energy-demanding,” Haining Zhu noted, suggesting, “you may have different insults in different diseases, but they all could lead to mitochondrial abnormality.”—Amber Dance.
Magrané J, Sahawneh MA, Przedborski S, Estévez AG, Manfredi G. Mitochondrial dynamics and bioenergetic dysfunction is associated with synaptic alterations in mutant SOD1 motor neurons. J Neurosci. 2012 Jan 4;32(1):229-42. Abstract