This is part 2 of a 2-part series on the neuromuscular junction in ALS. Check out part 1 here.
Alex Parker is delighted to see a potential ALS therapy, his Université de Montréal team and colleagues in Canada discovered in nematodes, reach the clinical-trial stage (Patten et al., 2017). 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.
There are certainly differences between species. A recent analysis reported that human NMJs are smaller, simpler, and more fragmented than those of rodents, yet remain stable over longer periods—indeed, they last the entire lifespan, while those of rodents degenerate in just a couple of years. Key NMJ proteins are also differentially expressed, with some at higher levels in people, and some higher in mice (Jones et al., 2017). Overall, though, the NMJs of vertebrates are “pretty similar,” said Steve Burden of the New York University Medical School.
An alternative approach, taken by University of Central Florida’s J Hickman and Xiufang Guo, is to build human ALS-specific NMJs in a dish and screen for drugs that stabilize them. They microfabricated chips with one side for iPSC-derived motor neurons and one for satellite cell-derived myotubes, with channels between them for axons to grow through. The two cell types connect and form functional NMJs that are able to transmit signals, causing the muscle cells to contract, according to a preliminary analysis presented at ALS/MND 2017 in Boston last December. Now, the team plans to use this system to identify potential therapies for ALS, in collaboration with Hesperos in Orlando, where Hickman is chief scientific officer.
Other scientists, too, are creating NMJs in vitro. At ALS/MND 2017, Brian Wainger’s laboratory at Massachusetts General Hospital in Boston presented its own model, also based on human iPSCs. And, Victor Rafuse’s team at Dalhousie University in Canada is building NMJs using mouse neurons and chick muscle cells (meeting abstracts IVT-08-IVT-10).
Going in vitro offers many advantages, said Hickman. His group can compare NMJs made with the cells from people with different forms of ALS, including, they plan, sporadic disease. “I think by having a direct human system, working on disease models, gives us a distinct advantage,” Hickman said.
Plus, this approach means Guo can connect disease-linked motor neurons with wild-type myotubes, and vice versa, to determine which cells are key to any NMJ deterioration. She can also apply the drug to only one cell type, to analyze which one benefits.
However, in vitro models have their downsides, too. They’re typically missing a crucial player: the perisynaptic Schwann cells that encircle the axons. “For a neuromuscular junction to be stable and function, to maintain and repair, they need perisynaptic Schwann cells,” said Virginia Tech’s Gregorio Valdez. (Other cell types could be added to NMJs-on-a-chip, Hickman and Guo said.) They also lack the blood supply, satellite cells, immune cells, and other features, Université de Montréal’s Richard Robitaille added.
Valdez and Burden pointed out other differences, too. For example, in animals the axon stops growing when it hits a muscle. In a dish, the axon often forms a junction, but keeps on growing past it, said Burden. And, since most in vitro models are only two-dimensional, they fail to replicate the three-dimensional structure of a proper junction, Valdez added.
“We’re getting closer, but we’re not there yet,” said Valdez. With further research, he suggested, it should be possible to solve these problems.
Burden said that current in vitro NMJs could still be a useful platform to screen for drugs, provided they pass a key test: There must be a clear difference between NMJs formed by wild-type cells and those carrying ALS-linked mutations. “As long as it replicates the detachment…then I’d say you have a good system,” he said.
As for Université de Montréal’s Pierre Drapeau, he said, “I still believe the whole animal readout is the best.” His collaborator, Justin Ichida at the University of Southern California in Los Angeles, tried pimozide out in their in vitro model and saw no effect. “It might be just be that they’re missing components that are in the animals, that aren’t in the dish,” Drapeau said.
Save the NMJs, Enough Said?
Scientists are also taking the more traditional, target-directed approach to find molecules that might protect NMJs (Jun 2017 news). Burden is hoping to bolster NMJs by boosting the activity of MuSK, a key enzyme that helps make these muscle-motor neuron connections. Increasing MuSK activity in ALS model mice delayed the start of muscle denervation by about 10 days, and decreased its extent for more than 40 days, enhancing muscle strength (Pérez-Garcia and Burden, 2012). Now, Burden’s team is developing a therapy using a similar strategy, by testing antibodies, developed by Genentech, that activate MuSK in these mice.
Meanwhile, Tivorsan Pharamceuticals in Providence, Rhode Island is testing biglycan, a small protein found in muscle extracellular matrix, as a potential treatment for ALS at the preclinical stage (Jun 2017 news). It plays a role in stabilizing the NMJ, likely by helping activate MuSK and regulating its density at the motor nerve terminals (Amenta et al., 2011).
Should one of these approaches keep muscles and motor neurons connected, what effects could they expect in people with ALS? “I’m not sure stabilizing NMJs would be sufficient, on its own, to ‘fix’ ALS, but it sure would help,” said Robitaille. Their muscles might stay stronger longer, he suggested.
It also might be possible to help rebuild damaged NMJs, Hickman suggested, perhaps with a second medication. “We think we might not only be able to halt progression, but also reverse the phenotype.”
What about survival? Increasing MuSK activity increased survival of ALS SOD1 model mice at most moderately (July 2017 news; Miyoshi et al., 2017). It’s not clear if preserving junctions would be enough. “Even if you only kept the synapses attached longer, that would in and of itself be a huge benefit, that’s my feeling,” said Burden.
The pimozide trial may offer some answers, but one thing is for certain: the NMJ is getting some much-deserved attention.
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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.