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.
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.
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.
Comments
Duke University
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.
University Medical Center Mainz
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.
Université Pierre et Marie Curie
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.
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