Scientists Call In The Chips To Model NMJs in ALS

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.

Dishing up NMJs. Researchers at the University of Central Florida coaxed iPS cells, from ALS patients, to form motor neurons and skeletal muscle cells. Then, they adhered them to a chip and linked them up to form functional NMJs in vitro. [Courtesy of Nadine Guo, University of Central Florida. Reproduced with permission.]

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.

PSCs not included. Perisynaptic (terminal) Schwann cells (PSCs) may become impaired in ALS suggesting that they too may need to be treated to rebuild and/or repair NMJs in the disease (Arbour et al., 2015; for review, Arbour et al., 2017) [Courtesy of Balice-Gordon, 1996, Current Biology, Cell Press.]

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.

Sugar, sugar. Biglycan (TVR-102) may help stabilize NMJs by localizing, and thereby helping activate MuSK at the motor neuron terminals. The potential drug, developed by Providence, Rhode Island startup Tivorsan Pharamceuticals, is approaching the clinic as a treatment for muscular dystrophy. [Courtesy of Brandan and Gutierrez, 2013. Matrix Biology. CC BY 4.0 license.]

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.

Featured Papers

Patten SA, Aggad D, Martinez J, Tremblay E, Petrillo J, Armstrong GA, La Fontaine A, Maios C, Liao M, Ciura S, Wen XY, Rafuse V, Ichida J, Zinman L, Julien JP, Kabashi E, Robitaille R, Korngut L, Parker JA, Drapeau P. Neuroleptics as therapeutic compounds stabilizing neuromuscular transmission in amyotrophic lateral sclerosis. JCI Insight. 2017 Nov 16;2(22). [PubMed]

Colón A, Guo X, Akanda N, Cai Y, Hickman JJ. Functional analysis of human intrafusal fiber innervation by human γ-motoneurons. Sci Rep. 2017 Dec 8;7(1):17202. [PubMed].

Guo X, Santhanam N , Kumanchik L , Long C, Wang Y, Thomas M, BaduMensah A, Colon A, Cai Y, Rumsey J, Shuler M, Hickman J. A human-based functional NMJ system for personalized ALS drug screening. Theme 2 In vitro experimental models. International Symposium on ALS/MND 2017. Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration 18:sup2, 106. [Meeting Abstract]


Jones RA, Harrison C, Eaton SL, Llavero Hurtado M, Graham LC, Alkhammash L, Oladiran OW, Gale A, Lamont DJ, Simpson H, Simmen MW, Soeller C, Wishart TM, Gillingwater TH. Cellular and molecular anatomy of the human neuromuscular junction. Cell Rep. 2017 Nov 28;21)9):2348-2356. [PubMed]

Devlin AC, Pereira J, Berezovski E, Sapir Y, Moakley D, Koh J, Wainger B. An all human iPSC neuromuscular junction (NMJ) model to link motor neuron (MN) excitability and neurodegeneration in ALS. Theme 2 In vitro experimental models. International Symposium on ALS/MND 2017. Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration 18:sup2, 106. [Meeting Abstract]

Rafuse E, Aubrey E, Leopold C, Rafuse V. An in vitro model of ALS to study chronic changes in function of the neuromuscular junction. Theme 2 In vitro experimental models. International Symposium on ALS/MND 2017. Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration 18:sup2, 106. [Meeting Abstract]

Miyoshi S, Tezuka T, Arimura S, Tomono T, Okada T, Yamanashi Y. DOK7 gene therapy enhances motor activity and life span in ALS model mice. EMBO Mol Med. 2017 Jul;9(7):880-889. [PubMed]

Amenta AR, Creely HE, Mercado ML, Hagiwara H, McKechnie BA, Lechner BE, Rossi SG, Wang Q, Owens RT, Marrero E, Mei L, Hoch W, Young MF, McChillan DJ, Rotundo RL, Fallon JR. Biglycan is an extracellular MuSK binding protein important for synapse stability. J Neurosci. 2012 Feb 15;32(7):2324-34. [PubMed]

Pérez-García MJ, Burden SJ. Increasing MuSK activity delays denervation and improves motor function in ALS mice. Cell Rep. 2012 Sep 27;2(3):497-502. [PubMed]

Amenta AR, Yilmaz A, Bogdanovich S, McKechnie BA, Abedi M, Khurana TS, Fallon JR. Biglycan recruits utrophin to the sarcolemma and counters dystrophic pathology in mdx mice. Proc Natl Acad Sci U S A. 2011 Jan 11;108(2):762-7. [PubMed]

Further Reading

Arbour D, Vande Velde C, Robitaille R. New perspectives on amyotrophic lateral sclerosis: the role of glial cells at the neuromuscular junction. J Physiol. 2017 Feb 1;595(3):647-661. [PubMed].

Thomson SR, Wishart TM, Patani R, Chandran S, Gillingwater TH. Using induced pluripotent stem cells (iPSC) to model human neuromuscular connectivity: promise or reality? J Anat. 2012 Feb;220(2):122-30. [PubMed].

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.

biglycan disease-als Dok7 MuSK neuromuscular junction NMJ topic-randd topic-researchmodels
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