Amyotrophic lateral sclerosis is a many-headed beast: Several genes can cause it, or no genes at all, and several cell types are involved beyond the motor neurons that actually degenerate. Scientists hoping to slay the monster still need to understand how different causes and pathologies lead to similar symptoms. Two papers this month offer some clues. A study in the August Archives of Neurology suggests that amyotrophic lateral sclerosis (ALS) falls into two basic categories: disease caused by mutations in the enzyme superoxide dismutase-1 (SOD1), and ALS due to any other cause, genetic or otherwise. Study leader Teepu Siddique, Northwestern University Feinberg School of Medicine in Chicago, Illinois, proposed that these two disparate causes lead to the same pathology. That common pathology, according to work published in the August 10 Nature Biotechnology online, involves toxic astrocytes. Researchers led by Brian Kaspar of Nationwide Children’s Research Institute in Columbus, Ohio, found that astrocytes derived from people with not only genetic, but also sporadic, ALS are bad news for motor neurons.

SOD1 Versus the Rest
Siddique’s group has been pursuing the hypothesis that SOD1 ALS fundamentally differs from other kinds. Non-SOD1 ALS encompasses familial cases with mutations in TAR DNA binding protein 43 (TDP-43), fused-in-sarcoma (FUS), and other mutations, some yet to be discovered. It also includes sporadic ALS, of unknown etiology, which may have an environmental basis. In recent work, the Siddique team showed that FUS is present in inclusions in spinal cords from many kinds of human ALS, but not the mutant SOD1 (mSOD1) version (see ARF related news story on Deng et al., 2010). First author Han-Xiang Deng led that and the current work in which the researchers pursued the role of another potential inclusion member, optineurin. This protein participates in a variety of cellular functions including intracellular trafficking and transcription activation.

In 2010, scientists in Japan discovered mutations in optineurin in some people with ALS (see ARF related news story on Maruyama et al., 2010). Optineurin mutations have also been linked to glaucoma. In sporadic ALS, optineurin protein showed up with TDP-43 in the skein-like inclusions characteristic of the disease. In familial ALS caused by the SOD1 mutation, optineurin co-localized with the dismutase in round inclusions. The authors suggested that optineurin inclusions could be a common feature among mSOD1 and non-mSOD1 ALS. But now, Siddique’s group concludes that optineurin does not co-localize with SOD1 inclusions.

Deng and colleagues collected 52 autopsy samples from people with sporadic ALS as well as familial ALS cases due to SOD1 and other mutations. They used the same antibodies as in the original Japanese study to compare optineurin staining patterns in these tissues with those from control samples.

Optineurin labeled skein-like inclusions in all 32 sporadic ALS cases, and in eight familial ALS cases without SOD1 mutations, including one with a TDP-43 mutation (the researchers also had a FUS case, but obtained too little material to test). Similarly, the Japanese team, led by Hideshi Kawakami of Hiroshima University, showed that FUS and TDP-43 show up in the majority of ALS inclusions (Ito et al., 2011; Ito et al., 2011). Thus, most ALS cases contain aggregates of TDP-43, FUS and optineurin.

The Hiroshima team also reported, in their 2010 paper, that optineurin co-localized with SOD1 inclusions. In contrast, the Northwestern researchers did not observe optineurin-positive inclusions in six SOD1 cases or in six controls. Similarly, the team could not find optineurin inclusions in samples from transgenic mice overexpressing mutant SOD1. The researchers concluded that SOD1-based ALS is a different sort of animal, with mSOD1 inclusions instead of the TDP-43-FUS-optineurin inclusions in other forms of familial and sporadic disease.

Why did Kawakami see optineurin with SOD1, while Siddique was unable to? “The differences between their and our results may be caused by different staining methods, different antibodies, or less optineurin in SOD1 ALS,” Kawakami wrote in an e-mail to ARF. “They stressed they used the same two antibodies we used, but we also used other antibodies for optineurin. Although SOD1 ALS may be different from other ALS, it is risky to depend only on optineurin staining.”

Yoshiaki Furukawa, who studies ALS proteins at the RIKEN Brain Science Institute in Wako City, Japan, was not involved in either study. He also pointed to antibody sensitivity as a possible explanation for the divergent results. Biochemical studies, looking for optineurin that is detergent-insoluble and thus likely in inclusions, could help settle the matter, he suggested in an e-mail to ARF.

Siddique conceded that his team might have missed optineurin in SOD1 inclusions, though he said, “If it is there, then it is in very small amounts.” Siddique’s team is now working to discover the mechanism of optineurin’s effects. In Kawakami’s paper, the authors suggested that mutant optineurin inhibits activation of the immunomodulator NF-κB, which has been linked to ALS before (Wang et al., 2011).

Siddique believes SOD1 mutations initiate a pathway distinct from other ALS forms. If SOD1 is mutated, it gloms together, excluding TDP-43, FUS, and optineurin. In contrast, those three proteins form aggregates in all other forms of ALS, where they exclude SOD1. Both kinds of aggregates contain ubiquitin and lead to motor neuron degeneration. “These two parallel pathways come together in the end,” he said.

Two Diseases United?
However, some studies suggest that SOD1 rears its head in sporadic ALS, too. Even without mutations, SOD1 can be unstable and aggregation prone (see ARF related news story on Banci et al., 2009 and ARF related news story on Nordlund et al., 2009), and one research group discovered misfolded SOD1 in some sporadic ALS cases (see ARF related news story on Bosco et al., 2010). Kaspar’s Nature Biotechnology paper suggests another commonality between SOD1 and other ALS forms.

Joint first authors Amanda Haidet-Phillips, Mark Hester, and Carlos Miranda were following up the work of others who discovered that mSOD1 motor neurons are stable when cultured alone, or when co-cultured with wild-type astrocytes, but perish at the hands of mSOD1 astrocytes (see ARF related news story on Nagai et al., 2007 and Di Giorgio et al., 2007). Most of those studies, however, relied not on wild-type, but astrocytes overexpressing mSOD1, at some 20 times beyond normal. The current work, Kaspar said, is the first to address the question with SOD1 at natural levels.

The researchers obtained autopsy samples from seven people who died of sporadic ALS, as well as one mSOD1 case and one non-ALS control. They were pleased to discover they could culture neuronal precursor cells from the tissues as late as three days postmortem. The scientists were able to differentiate these progenitors into astrocytes for their experiments.

Haidet-Phillips and colleagues added motor neurons derived from wild-type mouse stem cells to astrocyte cultures. Within four days of co-culture, the motor neurons exposed to mSOD1 or sporadic ALS astrocytes started to die. Motor neuron numbers plummeted 50 percent compared to co-cultures with control, non-ALS astrocytes. The degeneration was indistinguishable between the familial and sporadic cases, the authors reported. “We find it very intriguing and exciting that the progenitor cells either have a genetic risk factor, or retain a toxic phenotype they had in their original environment” Kaspar said.

In control experiments, the researchers determined that fibroblasts from people with ALS did not kill motor neurons, nor did ALS astrocytes damage GABAergic neurons. The effect seems to be particular to the astrocyte-motor neuron relationship. “I would not rule out that another cell might be toxic, but it appears that motor neurons are more susceptible [to astrocytes]” Kaspar said.

The ALS astrocytes were similarly toxic even when they did not directly contact motor neurons. Media from astrocyte cultures also killed the neurons. This work supports the idea, based on experiments with overexpressed SOD1, that astrocytes secrete what Kaspar called “a Darth Vader in the culture.”

To begin to seek out that dark molecular force responsible, the team examined gene expression among their astrocyte lines. They focused on inflammatory genes, which they knew were activated in mSOD1 astrocytes. Other researchers have fingered inflammatory molecules in mSOD1 mice (see ARF related news story on Di Giorgio et al., 2008 and Marchetto et al., 2008). Compared to non-ALS astrocytes, 84 inflammatory genes were upregulated in the ALS cells. Using cluster analysis, the scientists narrowed their focus to 22 genes that were most commonly upregulated in their samples. When the researchers used computer programs to analyze how these genes fit together into larger networks, they discovered that the top network involved included the NF-κB signaling complex—potentially linking astrocyte involvement in ALS to optineurin, a negative regulator of NF-κB (see Zhu et al., 2007).

Finally, the group wondered if they could temper astrocyte toxicity by knocking down SOD1 expression. In the case of the mutant SOD1 line, an SOD1-specific short hairpin RNA prevented any toxicity. In the case of sporadic ALS astrocyte lines, SOD1 knockdown was also protective, but not fully. The authors suggested that poor SOD1 knockdown explains the partial rescue in the sporadic ALS lines.

The progenitor-derived astrocytes will be a great tool for designing and screening therapeutics, wrote Christine Vande Velde of the University of Montréal, Canada, in an e-mail to ARF. “While this paper does not directly test the ‘misfolded SOD1 hypothesis,’ the siRNA experiments are quite strong in demonstrating that SOD1 itself is toxic in sporadic ALS,” she added. “I would speculate misfolded SOD1 conformers are the culprit.”

The results suggest that SOD1-targeted therapies might be applicable to a broad class of people with ALS, not just familial SOD1 cases. Isis Pharmaceuticals of Carlsbad, California, is currently testing a SOD1 antisense oligomer in people (see ARF related news story).—Amber Dance


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News Citations

  1. Where’s the FUS?—Evidence for Sporadic ALS Role Creates Stir
  2. Optineurin Mutations Cause ALS, If Not Glaucoma
  3. No Metal, No Stability: Structure of Apo SOD1
  4. Frustrated ALS Enzyme: SOD1 Sacrifices Structural Stability for Function
  5. Research Brief: SOD1 in Sporadic ALS Suggests Common Pathway
  6. Glia—Absolving Neurons of Motor Neuron Disease
  7. ALS in a Dish? Studying Motor Neurons from Human Stem Cells
  8. Clinical Trials for ALS: Taking Stock of 2009, Looking to 2010

Paper Citations

  1. . FUS-immunoreactive inclusions are a common feature in sporadic and non-SOD1 familial amyotrophic lateral sclerosis. Ann Neurol. 2010 Jun;67(6):739-48. PubMed.
  2. . Mutations of optineurin in amyotrophic lateral sclerosis. Nature. 2010 May 13;465(7295):223-6. PubMed.
  3. . Optineurin is co-localized with FUS in basophilic inclusions of ALS with FUS mutation and in basophilic inclusion body disease. Acta Neuropathol. 2011 Apr;121(4):555-7. PubMed.
  4. . Clinicopathologic study on an ALS family with a heterozygous E478G optineurin mutation. Acta Neuropathol. 2011 Aug;122(2):223-9. PubMed.
  5. . Activation of interferon signaling pathways in spinal cord astrocytes from an ALS mouse model. Glia. 2011 Jun;59(6):946-58. PubMed.
  6. . Structural and dynamic aspects related to oligomerization of apo SOD1 and its mutants. Proc Natl Acad Sci U S A. 2009 Apr 28;106(17):6980-5. PubMed.
  7. . Functional features cause misfolding of the ALS-provoking enzyme SOD1. Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9667-72. PubMed.
  8. . Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS. Nat Neurosci. 2010 Nov;13(11):1396-403. PubMed.
  9. . Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons. Nat Neurosci. 2007 May;10(5):615-22. PubMed.
  10. . Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nat Neurosci. 2007 May;10(5):608-14. PubMed.
  11. . Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation. Cell Stem Cell. 2008 Dec 4;3(6):637-48. PubMed.
  12. . Non-cell-autonomous effect of human SOD1 G37R astrocytes on motor neurons derived from human embryonic stem cells. Cell Stem Cell. 2008 Dec 4;3(6):649-57. PubMed.
  13. . Optineurin negatively regulates TNFalpha- induced NF-kappaB activation by competing with NEMO for ubiquitinated RIP. Curr Biol. 2007 Aug 21;17(16):1438-43. PubMed.

External Citations

  1. SOD1 antisense oligomer

Further Reading


  1. . Optineurin inclusions occur in a minority of TDP-43 positive ALS and FTLD-TDP cases and are rarely observed in other neurodegenerative disorders. Acta Neuropathol. 2011 Apr;121(4):519-27. PubMed.
  2. . Astrocyte loss of mutant SOD1 delays ALS disease onset and progression in G85R transgenic mice. Hum Mol Genet. 2011 Jan 15;20(2):286-93. PubMed.
  3. . An examination of wild-type SOD1 in modulating the toxicity and aggregation of ALS-associated mutant SOD1. Hum Mol Genet. 2010 Dec 15;19(24):4774-89. PubMed.
  4. . Astrocytic dysfunction: insights on the role in neurodegeneration. Brain Res Bull. 2009 Oct 28;80(4-5):224-32. PubMed.
  5. . Complexity of astrocyte-motor neuron interactions in amyotrophic lateral sclerosis. Neurodegener Dis. 2005;2(3-4):139-46. PubMed.
  6. . Aggregate formation in Cu,Zn superoxide dismutase-related proteins. J Biol Chem. 2003 Apr 18;278(16):14331-6. PubMed.

Primary Papers

  1. . Astrocytes from familial and sporadic ALS patients are toxic to motor neurons. Nat Biotechnol. 2011 Sep;29(9):824-8. PubMed.
  2. . Differential Involvement of Optineurin in Amyotrophic Lateral Sclerosis With or Without SOD1 Mutations. Arch Neurol. 2011 Aug;68(8):1057-61. PubMed.