Slacking off in ALS? Mutant SOD1 may partially close the SLACK ion channel resulting in increased excitability in some neurons (Zhang et al., 2017).[Image: NIGMS.]

Increased activity in the motor cortex of the brain may occur in most forms of ALS (see September 2015 news). But whether this hyperexcitability contributes to the disease remains an open question.

Now, researchers at Yale University make the case that ALS-linked mutant SOD1 may downregulate a key sodium-gated potassium ion channel, known as SLACK, through an apoptosis signal-regulatingkinase1 (ASK1)-based mechanism (Zhang et al., 2017).

The findings may help explain how motor neuron hyperexcitability occurs in ALS. These changes in excitability may contribute to disease pathogenesis and may underlie fasciculations, one of the earliest clinical manifestations of the disease.

The question is whether this pathway is the primary way that SOD1 mutations cause disease, said Steve Vucic of the University of Sydney, who was not involved in the study. If so, [there] is a tremendous opportunity for developing treatments against these kinase pathways.

The study is published on January 24 in the Journal of Neuroscience.

Excitement builds

Neuronal hyperexcitability emerged in recent years as an early and potentially unifying stepin ALS, due to its detection in a number of sporadic and genetic forms. While the evidence is still not yet conclusive, some studies suggest that this prolonged excitation can lead to toxicity, strengthening the case that these changes in excitability may contribute to the disease (Fritz et al., 2013; Hadzipasic et al., 2014).

How hyperexcitability occurs in ALS remains unclear. But a growing number of studies suggest that mutant SOD1 may be involved, at least in some cases of the disease (Wainger et al., 2014; van Zundert et al., 2008).

Researchers at Yale University, led by Leonard Kaczmarek and Arthur Horwich, wondered whether mutant SOD1 could trigger hyperexcitability in motor neurons by downregulating a key membrane-bound ion channel called SLACK (sequence like a calcium-activated K channel), also known as KCNT1 or KNa1.1.

SLACK is a key regulator of excitability that helps neuronsreturn to the resting state upon firing. Its widely expressed in the CNS and its dysfunction has also been implicated in neurological diseases including Fragile X and epilepsy (Barcia et al., 2012; Heron et al., 2012; Martin et al., 2014).

Hyperexcitability in the bag. Researchers use sea slug bag cell neurons to study underlying hyperexcitability mechanisms. [Image: Kabir et al., 2001 under a CC-BY-NC-SA license.]

To investigate this question, first co-authors Yalan Zhang and Weiming Ni turned to the neuronalmodel system, the sea slug Aplysia. The system gained recognition in the 1960s for its role in providing Eric Kandel Nobel Prize-winning insights into learning and memory formation.

The approachinvolves the manipulation and study of bag cell neurons, very large neuroendocrine cells in the sea slugs abdomen that control egg laying. The really big advantage is that, because of their size, you can inject materials into them and then use a very fine microelectrode to record changes in excitability, all without any disturbance of the cytoplasm, Kaczmarek said.

The researchers compared the activity of potassium channels in bag cell neurons in the presence or absence of wild-type or mutant SOD1, including soluble oligomers of increasing size. They found that SOD1 or mutant SOD1 G85R monomers had no effect. But when they injected SOD1 G85R oligomers, they observed a reduction in outward potassium currents by 20-30%. This drop occured within 10 minutes and increased with larger oligomer size.

Whats more, SOD1 G85R oligomers increased excitability of these neurons. Injection of these soluble 300 kDa protein complexes decreased the neurons resting membrane potential and increased its susceptibility to firing in response to applied stimuli, they found.

Further experiments identified the SLACK channel as the one most likely to have been affected by mutant SOD1, because neurons pretreated with siRNA against SLACK mitigated the effect of these protein complexes in these neurons.

Together, the results suggest that soluble mutant SOD1 oligomericcomplexes may lead to hyperexcitability due to partial closure of SLACK, a key sodium-gated potassium channel that helps neurons return to their resting state upon firing.

ASK1ing for trouble

How could mutant SOD1 downregulateSLACK? The researchers suspected that these effects may be triggered by ASK1, a key kinase that has been previously implicated in the destruction of motor neurons in the disease (Raoul et al., 2002).

ASK1 has been shown to mediate key effects of mutant SOD1 in mouse models of the disease including ER stress and disruption of axonal transport (Lee et al., 2016; Song et al., 2013). In addition, inhibitingthis pathway appears to extend the survival of a SOD1 G93A mouse model of the disease (Fujisawa, et al. 2016).

To investigate this possibility, the researchers blocked ASK1 signaling and determined the impact of SOD1 oligomeric complexes on potassium channel activity. They found that the suppression of outward potassium current could be abolished by pre-treatment with an inhibitor of the apoptosis signaling regulating kinase ASK1. Similar effects were achieved with an inhibitor of one of ASK1s downstream targets, JNK.

The results, Kaczmarek said, suggest that mutant SOD1 oligomericcomplexessuppressSLACK channels in neurons through a ASK1-based mechanism, causing hyperexcitability.

Its an attractive idea, says Massachusetts General Hospitals Brian Wainger, who was not involved in the study. The findings may provide a potentially direct mechanistic connection between mutant SOD1 and motor neuron hyperexcitability in ALS.

Mind your Potassium and KCNQs. Researchers are evaluating Kv 7.2 potassium channel activators including retigabine (orange) in hopes to reduce hyperexcitability in people with the disease. More specific channel modulators are being developed. One such activator, AUT00063, is being evaluated at the phase 2a stage by the London startup Autifony Therapeutics to treat hearing disorders. [Miceli et al., 2011 under CC BY 4.0 license.]

But a change in excitability may not be the only or even the most important consequence of SLACK down regulation, according to Kaczmarek. SLACK may act as an activity sensor, providing a direct link between neuronal firing and protein synthesis.

His teamhas previously shown that SLACK channel activity plays a role in synaptic development, through its ability to regulateactivity-dependent protein synthesis (Brown et al., 2010; Zhang et al., 2012). When you precipitate the channel from mammalian brain, it pulls down several messenger RNAs, he pointed out, and mutations that cause channel overactivity are associated with epilepsy (Barcia et al., 2012; Kim et al., 2015).

In fact, Kaczmarek added, it may not be the hyperexcitability of motor neurons that is toxic in ALS, but rather its proposed (but not yet tested) consequences on protein synthesis. A rapid change in the activity of these channels, as we saw here, is likely going to alter protein synthesis, and that can produce much longer-lasting effects, potentially more consistent with a late-onset disease.

This was an extremely elegant study, and an ingenious way to approach the issue of hyperexcitability, said Steve Vucic, who, in collaboration with University of Sydneys Matthew Kiernan in Australia helped identify these neuronal changes as an early sign of ALS in people with the disease. The goal now will be to see if this same pathway is affected in the mammalian models, or in human ALS iPS cells.

Brian Wainger agrees. The key questions, according to Wainger, are whether these findings hold up in mammalian models, and whether these findings can be generalized to other forms of the disease.

Searching for ALS-linked gene variants in SLACK or related ion channels might also provide insight into its relevance for the human disease, added Vucic.

Approaching the clinic

Hyperexcitability is clearly a clinical feature of many forms of familial and sporadic ALS, explains Wainger. Thats why it is attractive as a convergent mechanism for many forms of ALS. But one of the challenges is to determine to what extent an increase in firing is relevant for disease pathogenesis, rather than, as some argue, being a compensatory mechanism. Directly modulating excitability is one of the clearest ways of answering that question directly, he added.

If motor neuron hyperexcitabilitydoes hold up as a driver of disease, however, it may be a good target for therapy, according to Kaczmarek. I see this as very much a therapeutic possibility.

The reason is because opening up these potassium ion channels may help motor neurons in people with ALS return to their resting state and thereby, reduce hyperexcitability in the disease.

Finding magneto. Researchers are using transcranial magnetic stimulation to evaluate in part whether mexiletine and retigabine reduce hyperexcitability in people with the disease.[Image: NIH].

Kaczmareks team is now hoping to do just that by developing a SLACK activator. The project is ongoing.

In the meantime, clinicians are aiming to reduce hyperexcitability in people with ALS by repurposing existing medicines in hopes to treat the disease. Brian Wainger is leading an effort to determine whether the epilepsy drug retigabine may be helpful in ALS. The drug, identified by Wainger as a potential treatment while in the laboratory of Kevin Eggan, may help normalize the activity of motor neurons by opening up Kv7 potassium channels in people with the disease (see April 2016 news; ; Wainger et al., 2014).

Across the US, the University of Washingtons Michael Weiss is taking a different approach. He is evaluating whether mexiletine, a sodium channel blocker, may reduce hyperexcitability in people with the disease (see March 2016 news). Both strategies are currently at the phase 2 stage.

In a disease that has a selective neuronal vulnerability like ALS, says Wainger, I think it is likely that the electrophysiological properties of the neuron are going to be related to the degenerative nature of the disease. So normalizing those properties may have a good chance of being helpful.

References

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disease-als hyperexcitability mexiletine retigabine SOD1 topic-preclinical topic-researchmodels

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