Motor neurons, and methods to protect them, get much of the attention in ALS. But there’s something that goes wrong before motor neurons degenerate: their axons detach from muscle fibers, noted J. Hickman of the University of Central Florida (UCF) in Orlando: “We believe that the initial disease pathology occurs at the neuromuscular junction.”
That would explain why people with ALS gradually lose muscle strength, rather than immediately losing the ability to move, Hickman pointed out. It would also explain why potential therapies that reduced the loss of motor neurons in mice did not save the junctions, or the animals (Gould et al., 2006; Rouaux et al., 2007). And, once the neuromuscular junctions (NMJs) begin to crumble, muscle fibers, neurons and glia are likely in trouble too, added Gregorio Valdez of the Virginia Tech Carilion Research Institute in Roanoke.
These findings prompted Valdez, Hickman and others to focus their attention at the junction. Their groups are among those developing in vitro models of the NMJ, to better understand its workings and potentially screen for drugs that support these structures.
“This is something that’s sort of come back into vogue recently,” said Pierre Drapeau of the Université de Montréal in Canada. “We’re trying to catch things at the beginning.” Drapeau and his collaborators, screening in nematodes, happened upon a drug that seems to stabilize junctions (Patten et al., 2017). This medication, pimozide, is already approved for conditions such as schizophrenia. The researchers are now starting a phase 2b clinical trial.
One advantage of strengthening the NMJ is that it seems to collapse in all forms of ALS, noted UCF’s Xiufang (Nadine) Guo. While many scientists are developing treatments that target altered genes (Dec 2017 news), this approach might, potentially, help anyone with ALS, or other conditions where NMJs are affected, such as muscular dystrophy.
Of worms and fish, and mice and men
Drapeau wasn’t specifically looking for a NMJ stabilizer. In the wake of discoveries of TDP-43’s role in ALS (Oct 2006 news; Feb 2008 news), he started making zebrafish carrying human TDP-43 with disease-linked mutations. His colleague Alex Parker did the same with the worm Caenorhabditis elegans.
Typically, scientists go after a drug by identifying a specific molecular target, and then seek compounds that affect its function in vitro. But Drapeau and Parker didn’t know what TDP-43 was doing wrong in ALS. So, instead, they screened for small molecules with any benefit to their animals—drugs that would get the paralyzed worms and fish moving again.
Parker’s group screened nearly 4,000 small molecules, including clinically approved drugs, in the mutant TDP-43 nematodes. They found 24 compounds that restored mobility. Of those, the researchers considered 11 irrelevant—they were insecticides or other drug types that didn’t seem pertinent to the disease.
That left them with 13 hits, and all were neuroleptics: drugs such as tranquilizers that dampen nerve function. “That was this big surprise,” said Drapeau. “I didn’t believe it at first.”
Nonetheless, he treated his mutant TDP-43 zebrafish overnight with those drugs. Ten hits restored swimming, with pimozide providing the most benefit. “It was quite dramatic,” he said.
The researchers also tested pimozide on zebrafish models of FUS and SOD1 ALS. Again, it improved the speed, distance, and length of time the fish could swim, suggesting the drug might be of benefit to multiple forms of the disease.
The researchers suspected that the drug stabilized the NMJ in the fish by increasing neurotransmission (see Avila et al., 1989). They confirmed that by electrophysological analysis. Collaborator Richard Robitaille’s team, also at Université de Montréal, used a similar approach in mutant SOD1 mice. Again, pimozide improved neurotransmission.
How did pimozide do this? After all, it’s thought to work by blocking dopamine signaling in the brain. But, it’s no longer used for some conditions due to off-target side effects. Might blocking one of those unintended targets be helping stabilize the NMJs?
When Terry Snutch of the University of British Columbia in Vancouver, Canada, heard Drapeau speak about the findings, he offered a clue. His team found that pimozide can partially inhibit certain calcium channels (Santi et al., 2002). In the central nervous system, this enhances neurotransmitter release and strengthens the synapse, explained Robitaille. It’s not yet clear, however, if this happens at the NMJ, he added.
Encouraged by the results, the team turned to University of Calgary’s Lawrence Korngut in Canada to conduct a small phase 2, 6-week study to determine whether the approach is safe for people with ALS and could potentially be of benefit. 25 people with ALS participated.
Initially, the researchers aimed for a maximum dose of 6-10 milligrams per day, based on the typical dose of pimozide used clinically. However, this turned out to be difficult for people with ALS to tolerate. Korngut lowered the dose to 2-4 milligrams. Even this caused some weakness and restlessness.
To assess target engagement at the NMJs, the researchers used electromyography to measure the activity of 3 muscle pairs. Two were on either side of the hand, the third was in the upper back. They noted one significant change, in the right thumb: muscle function declined by less than 5% in participants taking pimozide compared to 5-15% in those taking placebo.
The researchers detected no significant difference in the rate of functional decline (ALS-FRS-R) or lung capacity. And, the placebo group progressed faster according to further analysis (Patten et al., 2017). Nonetheless, they took the change in the thumb muscle—one involved in fine motor control, and one of the earliest to degenerate in ALS—as a promising sign.
“We were very excited that Lawrence could see anything,” said Parker, “in six weeks.” Now, the researchers are embarking on a larger, double-blind, randomized, placebo-controlled study, with 100 people with ALS treated for up to 22 weeks.
“It’s a very important paper,” commented Guo, who was not involved in the work. “It highlights how important the NMJ is in the ALS disease.”
Kornegut, however, cautions that there is more work to do before we know whether pimozide may be of benefit to people with ALS. “We must first confirm that it is really useful and safe in the long term,” he said in a statement. “It is also important to be aware that pimozide is associated with significant side effects.” These include dizziness, blurred vision, and tremors.
Drapeau and Parker are developing pimozide derivatives that might have more focused activity, hoping to eliminate some of these unwanted side effects.
NMJs on Glass
Parker is delighted to see a project that started in nematodes reach the clinical-trial stage. But these worms, after all, are not human, and lack human NMJs. In fact, he recalled, some scientists told the researchers they were crazy to base a trial on these small-animal models.
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For people with ALS and their families, learn more about pimozide by checking out this recent ALS Canada talk presented by University of Calgary neurologist Lawrence Kornegut and the ALS Association blog.