In some familial forms of amyotrophic lateral sclerosis, mutant astrocytes damage neighboring neurons. Researchers do not know how. In the October 26 Nature Neuroscience online, scientists report a vital clue: Those mutant astrocytes are rife with a protein dimer comprising a sodium/potassium ATPase and an actin-binding protein. Blocking the ATPase protected neurons in culture and in mice. The results point to a potential therapeutic target for both familial and sporadic ALS. In fact, digoxin, a drug used to treat heart failure, blocks ATPases. However, digoxin at typical doses may not cross the blood-brain barrier efficiently enough to make a difference, pointed out senior author Azad Bonni of Washington University in St. Louis.

Co-culture of wild-type neurons (green) with mSOD1 astrocytes normally results in neurodegeneration (top). When researchers blocked 2-Na/K ATPase activity, the neurons survived (bottom). [Image courtesy of Nature Neuroscience]

First author Gilbert Gallardo and colleagues did not start out looking for an ALS treatment. They found the actin-binding protein when they tested an antibody for FOXO transcription factors on spinal cord lysates from mice carrying mutant human superoxide dismutase 1 (SOD1). “To our surprise, we saw a very strong band … but it was at the wrong size,” Bonni told Alzforum. The researchers wondered if it might be some kind of modified FOXO and decided to identify the protein. Bonni’s group has studied the role of these transcription factors in oxidative stress (Lehtinen et al., 2006).

In fact, mass spectrometry showed that the mystery band contained α-adducin, an actin-binding protein. Adducins cap actin filaments to control their length (Matsuoka et al., 2000). Using different antibodies to adducin, Gallardo and colleagues observed that both the protein and its phosphorylation were upregulated in mSOD1 mice once they reached 90 days old, which is just when ALS-like symptoms begin in these animals. At this point, Bonni and Gallardo suspected adducin might be poisoning the motor neurons of the sick mice. Immunohistochemistry brought another surprise: The α-adducin co-localized with a marker for astrocytes, not neurons.

As Gallardo was performing these experiments, researchers in the ALS field were also focusing their attention on astrocytes. Several studies indicated that mSOD1-toting astrocytes, and even astrocytes from people with sporadic ALS, release some factor that damages wild-type motor neurons, (see Apr 2007 news story; Feb 2014 news story). Researchers have not yet figured out what that substance might be or what promotes its release. Bonni realized they might have stumbled onto part of the answer.

To investigate further, they co-cultured astrocytes expressing mSOD1 with wild-type primary spinal motor neurons. As other researchers have reported, the mutant astrocytes killed the neurons. Treating the astrocytes with interfering RNA to knock down α-adducin blocked the neurotoxicity. Gallardo saw the same protection in mSOD1 mice. When he injected a lentivirus with α-adducin-interfering RNA into one side of the lumbar spinal cord of the animals, more motor neurons survived than on the untreated side of the cord.

To better understand how α-adducin contributes to neurotoxicity, the authors looked for binding partners. They immunoprecipitated adducin from mSOD1 mice and used mass spectrometry to identify proteins that came along for the ride. One partner was the α2-Na/K ATPase. Previous research has indicated that adducins might activate these ion pumps in the kidney (Ferrandi et al., 1999). Astrocytes in mSOD1 mice overproduced the α2-Na/K ATPase, as they did α-adducin. Knockdown of the ATPase in these astrocytes protected motor neurons in co-cultures and in mice.

Bonni was excited by these findings because he knew of approved drugs that suppress the activity of these ATPases. The most commonly used is digoxin, which treats heart failure by improving the muscle’s ability to contract. Treating the mSOD1 astrocyte and neuron co-cultures with digoxin rescued the neurons.

The authors do not know if digoxin will be protective in mSOD1 mice, but they have crossed those animals with mice that make half the normal amount of α2-Na/K ATPase. SOD1 mice with reduced α2-Na/K ATPase survived 20 days longer than their littermates with normal ATPase levels.

To see if people with ALS also overproduce the ATPase, Gallardo and colleagues examined tissue from people who had died from sporadic disease or from familial ALS due to SOD1 mutations. Spinal cord lysates from both groups contained more α-adducin and α2-Na/K ATPase than normal lysates. Bonni said it would be worthwhile to examine lysates from people with other familial forms caused by other genes, such as TDP-43 and C9ORF72. He is also interested in looking at samples from people who had other neurodegenerative diseases where glia are suspected of causing damage, such as Huntington’s (Faideau et al., 2010; Shin et al., 2009).

Bonni has received inquiries from ALS patients eager to try digoxin. He cautioned that if digoxin were taken orally at the usual dosage, sufficient amounts might not reach the spinal cord, while higher doses could cause serious side effects, including vomiting and difficulty breathing. The next step, he said, would be to treat ALS model mice by placing digoxin directly into the central nervous system. “If that works, there might be a possibility of going into clinical trials,” Bonni speculated. In the long run, he added, it would be preferable to develop a drug specific for the α2 version of the ATPase. He said ideally, that would be a pill that could be taken orally. Another option would be a biologic, such as a small interfering RNA, that would have to be provided directly to the CNS.

Scientists said that while the findings are important, questions remain as to how astrocytes kill motor neurons (see comments below). “This paper has revealed an important and powerful new molecular player … but the hunt for the actual toxic molecule is still on!” wrote Christian Lobsiger of the INSERM Brain and Spinal Cord Institute in Paris. Researchers cannot yet explain how mutant SOD1 boosts α-adducin or α2-Na/K ATPase expression in the astrocytes, nor can they describe how those proteins cause the cells to deliver the death blow to motor neurons.

Bonni and colleagues suggested a possible mechanism. An excess of ATPase, they theorized, might increase the pressure on mitochondria to keep up with the enzyme’s demand for ATP. Indeed, they observed unusually high oxygen uptake by mSOD1 astrocytes, indicating the mitochondria were particularly active. Overworked mitochondria might produce reactive oxygen species, turning on inflammation in those astrocytes and causing them to secrete cytokines and other factors that could harm neurons, the researchers speculate. Supporting their hypothesis, Gallardo and colleagues found that production of several inflammatory molecules was increased in mSOD1 astrocytes, but downregulated in astrocytes from the mSOD1 mice that were heterozygous for α2-Na/K ATPase.—Amber Dance


  1. To my knowledge, this is the first time this particular mechanism has been manipulated in any model of ALS. While this preclinical data is very interesting, it is not clear that it means anything for people with ALS yet. Many other drugs prolong survival in cell cultures and animals with ALS-causing mutations, but only one has ever worked to some degree in people (riluzole). Couple this dramatic history of translation failures with the many side effects and drug-drug interactions of digitalis, and it is clear that more work needs to be done before digitalis can be recommended as an ALS treatment. A reasonable next step might be a small, carefully monitored safety trial.

    View all comments by Richard Bedlack
  2. The study by Gallardo and colleagues is certainly a significant and important contribution to the field. The authors provide compelling evidence, by cell culture and genetic studies as well as by a pharmacological treatment, that a new player in the field, the α-adducin/α2-Na/K ATPase complex, is of critical relevance for the pathogenesis of SOD1-mediated ALS. They thereby provide an explanation for the non-cell autonomous mechanism of the disease, which is at least partially mediated by astrocytes. More specifically, the upregulation of the complex and the subsequent increase of Na/K ATPase activity in astrocytes substantially contribute to the toxicity of mutant SOD1 astrocytes in co-culture experiments and to the fast progression of disease in an ALS mouse model. Alpha-adducin and α2-Na/K ATPase are also upregulated in spinal cords from ALS patients. The inhibition of the Na/K-ATPase activity by the ATPase blocker digoxin is protective, at least in the co-culture model. Despite the intriguing findings, some questions concerning a potential treatment as well as the underlying cell biology are not resolved.

    Given the fact that the upregulation of α2-Na/K ATPase in astrocytes is relevant for the disease and that a specific inhibitor might efficiently inhibit α2-Na/K ATPase activity in the central nervous system, it still remains unclear how microglia contribute to the disease progression under these conditions. It has been shown by several labs that besides astrocytes, microglia are also critical for the progression of ALs. At least morphologically, microglia activation and microglia numbers are not changed upon altering astrocytic Na/K ATPase levels/activity.

    There is indeed compelling evidence that Na/K ATPase activity is critical for making mutant SOD1-expressing astrocytes bad actors. It would certainly be of interest to investigate how mutant SOD1 triggers the expression of α-adducin and α2-Na/K ATPase. The initial experiment that resulted in the identification of α-adducin was a screen with an antibody recognizing phosphorylation events after oxidative stress. Having this initial experiment in mind, increased oxidative stress might be the critical factor to initiate the detrimental cascades. However, the molecular basis for how increased α2-Na/K ATPase activity in astrocytes results in a conditioned medium that is toxic to motor neurons is still elusive.

    View all comments by Albrecht Clement
  3. This study by Gallardo et al. from the Bonni lab is an impressive work that reveals essential new insights into the enigmatic glial-derived non-cell autonomous toxicity of ALS.

    The paper starts with a classic scientific approach—simple and unspectacular—the authors were looking for phosphorylation events in the spinal cords of symptomatic mutant SOD1 ALS mice. Unexpectedly, as they state themselves, they found a protein called “α-adducin” to be strongly increased and phosphorylated in affected spinal cord lysates. They took their chances and tested if this protein could be implicated in ALS disease mechanism—and struck gold.

    Phosphorylated α-adducin, a protein involved in actin filament stability, seems to be specifically increased in astrocytes during the disease in ALS mice—this gave them the hint that they could be tracking astroglial-derived ALS toxicity. Indeed, RNAi-mediated knockdown of α-adducin in mouse primary ALS astrocytes reduced their known toxicity toward primary mouse motor neurons. Even more convincingly, by using an elaborate lentiviral approach to locally downregulate α-adducin in vivo in spinal cord ventral horn astrocytes, they were able to demonstrate reduced motor-neuron degeneration. However, the real question was how increased phosphorylated α-adducin could become toxic for motor neurons.

    Using immunoprecipitation, they found a specific Na/K-ATPase to be associated with α-adducin in symptomatic ALS mouse spinal cords. Again, and unexpectedly, this α2-Na/K-ATPase was quite specific for astrocytes and induced in spinal cords of ALS mice. Using the same strategy as for α-adducin, they demonstrated that knockdown of this Na/K-ATPase form, either in primary ALS astrocytes or in vivo using their impressive lentiviral approach, was able to reduce astroglial-derived ALS neurotoxicity.

    In a final in vivo experiment, they crossed mice with heterozygous deletions of this Na/K-ATPase form with mutant SOD1 ALS mice, and could clearly reveal a neuroprotective action and survival expanding effect on the crosses.

    The next question is obviously now to exactly define the toxic agent that comes from the ALS astrocytes and figure out how is it linked to a Na/K-pump. The paper gives first hints: Knockdown of the Na/K-ATPase in primary ALS astrocytes leads to reduction of different secreted inflammatory molecules. In addition, it seems that increased Na/K-ATPase levels increase mitochondrial respiration. Thus in ALS astrocytes, it is beneficial to tune down (possibly hyperactive) mitochondrial respiration, and by this, most likely produce less neurotoxic ROS components.

    However, is the effect of increased α-adducin/Na/K-ATPase really linked to mutant SOD1 action in astrocytes, or more to a general disease-linked neurotoxic action of activated astrocytes (independent of mutant SOD1)? Indeed, increased α-adducin/Na/K-ATPase levels could be found in both familial (SOD1) and sporadic ALS patient spinal cord lysates. Likewise, although the data for selective astrocyte expression and induction of both phospho-α-adducin and the α2-Na/K-ATPase are quite convincing, trace expressions in other glial/neuronal cell populations could nevertheless contribute to the toxic effect.

    The paper has revealed an important and powerful new molecular player in the search for the identity of non-cell autonomous glial-derived ALS toxicity—but the hunt for the actual toxic molecule is still on! On the other hand, as pharmacological blockage of specific Na/K-ATPase forms is widely used to treat heart failure, new perspectives for ALS patients might really open up.

    View all comments by Christian Lobsiger

Make a Comment

To make a comment you must login or register.


News Citations

  1. Glia—Absolving Neurons of Motor Neuron Disease
  2. Death in a Dish: Astrocytes from ALS Patients Flick Necroptosis Switch in Motor Neurons

Paper Citations

  1. . A conserved MST-FOXO signaling pathway mediates oxidative-stress responses and extends life span. Cell. 2006 Jun 2;125(5):987-1001. PubMed.
  2. . Adducin: structure, function and regulation. Cell Mol Life Sci. 2000 Jun;57(6):884-95. PubMed.
  3. . Evidence for an interaction between adducin and Na(+)-K(+)-ATPase: relation to genetic hypertension. Am J Physiol. 1999 Oct;277(4 Pt 2):H1338-49. PubMed.
  4. . In vivo expression of polyglutamine-expanded huntingtin by mouse striatal astrocytes impairs glutamate transport: a correlation with Huntington's disease subjects. Hum Mol Genet. 2010 Aug 1;19(15):3053-67. Epub 2010 May 21 PubMed.
  5. . Expression of mutant huntingtin in mouse brain astrocytes causes age-dependent neurological symptoms. Proc Natl Acad Sci U S A. 2009 Dec 29;106(52):22480-5. PubMed.

External Citations

  1. SOD1

Further Reading


  1. . Glial cells as intrinsic components of non-cell-autonomous neurodegenerative disease. Nat Neurosci. 2007 Nov;10(11):1355-60. PubMed.
  2. . Activation of interferon signaling pathways in spinal cord astrocytes from an ALS mouse model. Glia. 2011 Jun;59(6):946-58. PubMed.
  3. . The non-cell-autonomous component of ALS: new in vitro models and future challenges. Biochem Soc Trans. 2014 Oct;42(5):1270-4. PubMed.
  4. . Motor neuron death in ALS: programmed by astrocytes?. Neuron. 2014 Mar 5;81(5):961-3. PubMed.
  5. . Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS. Proc Natl Acad Sci U S A. 2014 Jan 14;111(2):829-32. Epub 2013 Dec 30 PubMed.
  6. . Astrocytes expressing mutant SOD1 and TDP43 trigger motoneuron death that is mediated via sodium channels and nitroxidative stress. Front Cell Neurosci. 2014;8:24. Epub 2014 Feb 7 PubMed.
  7. . Expression of ALS-linked TDP-43 mutant in astrocytes causes non-cell-autonomous motor neuron death in rats. EMBO J. 2013 Jul 3;32(13):1917-26. PubMed.

Primary Papers

  1. . An α2-Na/K ATPase/α-adducin complex in astrocytes triggers non-cell autonomous neurodegeneration. Nat Neurosci. 2014 Dec;17(12):1710-9. Epub 2014 Oct 26 PubMed.