4 November 2011. If you have wondered about the true importance of mitochondria in motor neuron disease, read on. Researchers from the Weill Medical College of Cornell University in New York corralled mutant superoxide dismutase 1 (mSOD1), a ubiquitous protein that causes amyotrophic lateral sclerosis (ALS), in these powerhouse organelles to prove that mSOD1 damages the mitochondria, and in turn, the cell and the body as a whole. In the November 2 Journal of Neuroscience, the researchers report that limiting the dismutase to the mitochondrial intermembrane space is sufficient to recapitulate much, but not all, of the ALS pathology caused by the SOD1 mutation, which causes a rare form of inherited ALS.
There is plenty of evidence that mitochondria play a part in the motor neuron degeneration that happens in ALS (reviewed in Hervias et al., 2006), but so do defects in many other areas such as RNA processing (see ARF related news story on Kwiatkowski et al., 2009 and Vance et al., 2009), the endoplasmic reticulum (see ARF related news story on Saxena et al., 2009) and Golgi (Urushitani et al., 2008), neighboring glia (reviewed in Ilieva et al., 2009), glutamate uptake (Rothstein et al., 1995), and axonal transport (see ARF related news story). The study authors, led by joint first authors Anissa Igoudjil and Jordi Magrané and senior author Giovanni Manfredi, sought to delineate the cause and outcomes of only the mitochondrial dysfunction. Researchers wondered whether malformed, underactive mitochondria cause some or much of the widespread pathology in ALS—or whether they are simply a symptom of a struggling cell. The current work confirms, as Manfredi’s team and others had also seen in cultured cells (Magrané et al., 2009; Cozzolino et al., 2009), that mSOD1 directly influences mitochondria for the worse, even if all else in the cell is normal.
The researchers designed a mouse model that makes the G93A mutant human SOD1 (an alanine for glycine at position 93), under control of the prion promoter. They linked the dismutase to the amino terminus of mitofilin, which targeted it to the inner mitochondrial membrane, facing the intermembrane space. The mitochondria of these mito-mSOD1 mice contained about the same amount of mSOD1 seen in mitochondria in the standard SOD1-G93A model with unrestricted mSOD1 targeting.
Mice with pan-cellular SOD1-G93A succumb to disease within a year. Male mice with mitochondrial mSOD1, in contrast, lived a nearly normal lifespan of 18 months, Manfredi said. Females suffered more severe disease, and their illness required they be sacrificed for ethical reasons by one year. That may be because the transgene landed in an estrogen-sensitive locus, not due to the mitochondrial mSOD1, Manfredi said. Males and females aged prematurely, hunching over and losing weight before their time. Compared to non-transgenic mice of either gender, females struggled with the motor coordination rotarod test when first examined, at three months of age, while males had near-normal coordination until six months. On a hang test for muscle strength, females performed worse than non-transgenics starting at three months, while males’ muscle weakness was not statistically significant.
When the researchers examined the mitochondria from the mito-mSOD1 mice under the electron microscope, they observed large, empty spaces, or vacuoles, in the normally densely packed organelles. In biochemistry experiments, mitochondria isolated from the brains of mito-mSOD1 mice were more sensitive to an uncoupling agent, failed to retain calcium ions, and had reduced activity of the respiratory enzyme cytochrome oxidase, as compared to mitochondria from non-transgenic mice. These data indicate that while the mitochondria with mutant SOD1 were functional, they were weakened and easily ran out of energy when stressed. Manfredi compared them to a four-cylinder engine firing on only three.
The effects of the mitochondrial mSOD1 extended beyond that organelle.
Compared to normal mice, fewer motor neurons populated the spinal cord of mito-mSOD1 mice, and they suffered a thinning of the motor cortex. The motor neuron loss was not as bad as in standard SOD1-G93A mice, the authors noted. In addition, one hallmark of ALS was decidedly missing: “What was surprising to us is, despite the fact that the mice lost a proportion of the spinal cord neurons and there was atrophy of skeletal muscle, we did not see denervation,” Manfredi said. He noted that just because neuromuscular junctions were intact, it does not mean they were healthy—the sickened neurons could still fail to send proper signals through the junction without completely detaching from it, explaining the poor rotarod performance.
The researchers concluded that mitochondrial mSOD1 is only responsible for part of ALS pathology. “It is now becoming clear that a combination of toxic effects are probably necessary to drive motor neuron disease onset and progression,” wrote Adrian Israelson of the University of San Diego, who was not involved in the study, in an e-mail to ARF.
Precisely how mSOD1 disables mitochondria is unknown. It might interact with proteins or other factors in the respiratory chain, Manfredi suggested, or it might promote the formation of free radicals. The new mito-mSOD1 mouse can help answer that question, commented Piera Pasinelli of Thomas Jefferson University in Philadelphia, Pennsylvania. “This is a powerful tool to really dissect the specific contribution for the mutant SOD1 in these organelles,” said Pasinelli, who also was not involved in the current paper.
One pathological event that is relevant to mSOD1 in mitochondria is production of reactive oxygen species, which generate other toxins, such as peroxynitrite, which can drive apoptosis, or programmed cell death. SOD chemically modifies other proteins in the presence of peroxynitrite. In the October 27 Journal of Biological Chemistry online, researchers from the University of Melbourne, Australia, propose a potential treatment for this mSOD1 effect. They discovered that a copper compound that scavenges peroxynitrite extended lifespan, and reduced inflammation, in a mouse model for ALS that expresses lower levels of SOD1-G93A than Manfredi’s mice. “They do not look at mitochondria directly, but it is possible that there is an effect of the drug at the mitochondrial level,” Manfredi wrote in an e-mail to ARF. In addition, Manfredi noted, this study reports for the first time that their low-expressing G93A mice also exhibit TAR DNA Binding Protein 43 (TDP-43) pathology, which up until now had not been seen in mSOD1 mice. TDP-43 accumulated as fragmented, abnormally phosphorylated protein in the spinal cord of these animals. The study was led by first author Cynthia Soon and senior authors Kevin Barnham and Qiao-Xin Li.—Amber Dance.
Igoudjil A, Magrané J, Fischer LR, Kim HJ, Hervias I, Dumont M, Cortez C, Glass JD, Starkov AA, Manfredi G. In vivo pathogenic role of mutant SOD1 localized in the mitochondrial intermembrane space. J Neurosci. 2011 Nov 2;31(44):15826-15837. Abstract
Soon CP, Donnelly PS, Turner BJ, Hung LW, Crouch PJ, Sherratt NA, Tan JL, Lim NKH, Lam L, Bica L, Lim SC, Hickey JL, Morizzi J, Powell A, Finkelstein DK, Culvenor JG, Masters CL, Duce J, White AR, Barnham KJ, Li QX. CUII(ATSM) protects against peroxynitrite-induced nitrosative damage and prolonged survival in amyotrophic lateral sclerosis mouse model. J Biol Chem. 2011 Oct 27. Abstract