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-regulating kinase 1 (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.
Neuronal hyperexcitability emerged in recent years as an early and potentially unifying step in 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 neurons return 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).
To investigate this question, first co-authors Yalan Zhang and Weiming Ni turned to the neuronal model 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 approach involves the manipulation and study of bag cell neurons, very large neuroendocrine cells in the sea slug’s 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.
What’s more, SOD1 G85R oligomers increased excitability of these neurons. Injection of these soluble 300 kDa protein complexes decreased the neuron’s 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 oligomeric complexes 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 downregulate SLACK? 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, inhibiting this 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 ASK1’s downstream targets, JNK.
The results, Kaczmarek said, suggest that mutant SOD1 oligomeric complexes suppress SLACK channels in neurons through a ASK1-based mechanism, causing hyperexcitability.
“It’s an attractive idea,” says Massachusetts General Hospital’s 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.
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 team has previously shown that SLACK channel activity plays a role in synaptic development, through its ability to regulate activity-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 Sydney’s 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. That’s 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 hyperexcitability does 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.
Kaczmarek’s 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 Washington’s 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.”
Zhang Y, Ni W, Horwich AL, Kaczmarek LK. An ALS-associated mutant SOD1 rapidly suppresses KCNT1 (Slack) Na+-activated K+ channels in Aplysia neurons. J Neurosci. 2017 Jan 24. pii: 3102-16. [PubMed]
Fritz E, Izaurieta P, Weiss A, Mir FR, Rojas P, Gonzalez D, Rojas F, Brown RH Jr, Madrid R, van Zundert B. Mutant SOD1-expressing astrocytes release toxic factors that trigger motoneuron death by inducing hyperexcitability. J Neurophysiol. 2013 Jun;109(11):2803-14. 2013 Mar 13. [PubMed].
Hadzipasic M, Tahvildari B, Nagy M, Bian M, Horwich AL, McCormick DA. Selective degeneration of a physiological subtype of spinal motor neuron in mice with SOD1-linked ALS. Proc Natl Acad Sci U S A. 2014 Nov 25;111(47):16883-8. [PubMed].
Wainger BJ, Kiskinis E, Mellin C, Wiskow O, Han SS, Sandoe J, Perez NP, Williams LA, Lee S, Boulting G, Berry JD, Brown RH Jr, Cudkowicz ME, Bean BP, Eggan K, Woolf CJ. Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons. Cell Rep. 2014 Apr 10;7(1):1-11. [PubMed]
van Zundert B, Peuscher MH, Hynynen M, Chen A, Neve RL, Brown RH Jr, Constantine-Paton M, Bellingham MC. Neonatal neuronal circuitry shows hyperexcitable disturbance in a mouse model of the adult-onset neurodegenerative disease amyotrophic lateral sclerosis. J Neurosci. 2008 Oct 22;28(43):10864-74. [PubMed].
Barcia G, Fleming MR, Deligniere A, Gazula VR, Brown MR, Langouet M, Chen H, Kronengold J, Abhyankar A, Cilio R, Nitschke P, Kaminska A, Boddaert N, Casanova JL, Desguerre I, Munnich A, Dulac O, Kaczmarek LK, Colleaux L, Nabbout R. De novo gain-of-function KCNT1 channel mutations cause malignant migrating partial seizures of infancy. Nat Genet. 2012 Nov;44(11):1255-9. [PubMed].
Heron SE, Smith KR, Bahlo M, Nobili L, Kahana E, Licchetta L, Oliver KL, Mazarib A, Afawi Z, Korczyn A, Plazzi G, Petrou S, Berkovic SF, Scheffer IE, Dibbens LM. Missense mutations in the sodium-gated potassium channel gene KCNT1 cause severe autosomal dominant nocturnal frontal lobe epilepsy. Nat Genet. 2012 Nov;44(11):1188-90. [PubMed].
Martin HC, Kim GE, Pagnamenta AT, Murakami Y, Carvill GL, Meyer E, Copley RR, Rimmer A, Barcia G, Fleming MR, Kronengold J, Brown MR, Hudspith KA, Broxholme J, Kanapin A, Cazier JB, Kinoshita T, Nabbout R; WGS500 Consortium., Bentley D, McVean G, Heavin S, Zaiwalla Z, McShane T, Mefford HC, Shears D, Stewart H, Kurian MA, Scheffer IE, Blair E, Donnelly P, Kaczmarek LK, Taylor JC. Clinical whole-genome sequencing in severe early-onset epilepsy reveals new genes and improves molecular diagnosis. Hum Mol Genet. 2014 Jun 15;23(12):3200-11. [PubMed].
Raoul C, Estévez AG, Nishimune H, Cleveland DW, deLapeyrière O, Henderson CE, Haase G, Pettmann B. Motoneuron death triggered by a specific pathway downstream of Fas. potentiation by ALS-linked SOD1 mutations. Neuron. 2002 Sep 12;35(6):1067-83. [PubMed].
Lee S, Shang Y, Redmond SA, Urisman A, Tang AA, Li KH, Burlingame AL, Pak RA, Jovičić A, Gitler AD, Wang J, Gray NS, Seeley WW, Siddique T, Bigio EH, Lee VM, Trojanowski JQ, Chan JR, Huang EJ. Activation of HIPK2 Promotes ER Stress-Mediated Neurodegeneration in Amyotrophic Lateral Sclerosis. Neuron. 2016 Jul 6;91(1):41-55. [PubMed].
Song Y, Nagy M, Ni W, Tyagi NK, Fenton WA, López-Giráldez F, Overton JD, Horwich AL, Brady ST. Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm. Proc Natl Acad Sci U S A. 2013 Apr 2;110(14):5428-33. [PubMed].
Fujisawa T, Takahashi M, Tsukamoto Y, Yamaguchi N, Nakoji M, Endo M, Kodaira H, Hayashi Y, Nishitoh H, Naguro I, Homma K, Ichijo H. The ASK1-specific inhibitors K811 and K812 prolong survival in a mouse model of amyotrophic lateral sclerosis. Hum Mol Genet. 2016 Jan 15;25(2):245-53. [PubMed].
Brown MR, Kronengold J, Gazula VR, Chen Y, Strumbos JG, Sigworth FJ, Navaratnam D, Kaczmarek LK. Fragile X mental retardation protein controls gating of the sodium-activated potassium channel Slack. Nat Neurosci. 2010 Jul;13(7):819-21. [PubMed].
Zhang Y, Brown MR, Hyland C, Chen Y, Kronengold J, Fleming MR, Kohn AB, Moroz LL, Kaczmarek LK. Regulation of neuronal excitability by interaction of fragile X mental retardation protein with slack potassium channels. J Neurosci. 2012 Oct 31;32(44):15318-27. [PubMed].
Kim GE, Kronengold J, Barcia G, Quraishi IH, Martin HC, Blair E, Taylor JC, Dulac O, Colleaux L, Nabbout R, Kaczmarek LK. Human slack potassium channel mutations increase positive cooperativity between individual channels. Cell Rep. 2014 Dec 11;9(5):1661-72. [PubMed].