25 January 2013. A dearth of calcium appears to silence nerves attempting to communicate with muscles in a zebrafish model expressing a mutant version of the amyotrophic lateral sclerosis gene TDP-43. Because of this, scientists propose, motor neurons shrivel away from their muscular connections. Drugs that open up calcium channels in those neurons restore both this communication and the fishes' ability to swim properly. The study appears in the January 23 Journal of Neuroscience. Similar medicines might benefit people in the early stages of ALS, suggested first author Gary Armstrong, who performed the study in the laboratory of Pierre Drapeau at the University of Montréal in Canada.
Armstrong and Drapeau studied the mechanisms of TDP-43 pathology in zebrafish larvae because their neurons make it possible to examine hundreds of fish in easy patch-clamp studies in one day. The experiments would have been much more complicated in a mammal, Armstrong told Alzforum. He worked on non-transgenic fish and on others expressing either wild-type human TDP-43 or a glycine-368-cysteine mutation found in human ALS (Kühnlein et al., 2008). The larvae expressing wild-type TDP-43 were fairly normal, but the mutants swam slowly and only for short distances when nudged (Kabashi et al., 2010).
To find out what crippled the fish, Armstrong examined how their motor neurons and muscles handled action potentials. Individually, each cell type carried current well, although the motor neurons of the mutant fish were easy to overexcite. The defect became clear when Armstrong provided current to neurons and recorded the resulting signal, called endplate current, in the adjacent muscle cells. “There was poor communication, and when they did communicate, these endplate currents were reduced,” Armstrong said. “That suggests there is something wrong … at the neuromuscular junction.”
A healthy junction maintains itself by way of regular signaling. Armstrong theorized that the faulty communication, over time, could lead to the dying back of axons from the synapse. Such pulling back is among the earliest events known in ALS, both in people and in animal models; it is detectable even before symptoms arise (Fischer et al., 2004; Murray et al., 2010; Dadon-Nachum et al., 2011). Muscle endplates become denervated early in disease (see Maselli et al., 1993) and in rodents expressing mutant TDP-43 or superoxide dismutase 1, another ALS gene (Zhou et al., 2010; Swarup et al., 2011; Frey et al., 2000).
What might go wrong at the neuromuscular junction of fish with mutant TDP-43? In frogs, calcium channels are important players in the release of acetylcholine at that junction. In response to an action potential, calcium rushes into axon tips, setting loose the neurotransmitter, Armstrong said (Thaler et al., 2001; Arenson and Evans, 2001; Nurullin et al., 2011).
Trying various drugs to affect the calcium channels in the fish, Armstrong discovered that opening L-type voltage-dependent calcium channels restored movement. The experimental channel agonists FPL 64176 and Bay K 8644 both returned swimming speed and distance to normal in the mutant fish. Neither of these compounds has been approved for human use. In patch-clamp experiments, Armstrong found that the agonists returned neuron-muscle communication back to normal. He suspects that opening up the channels leads to greater calcium influx during action potentials. “That allows for a greater number of these acetylcholine vesicles to be … dumped into the neuromuscular junction,” he said. In an e-mail to Alzforum, Hongxia Zhou of Thomas Jefferson University in Philadelphia, Pennsylvania, commented that “the study provides one more piece of evidence supporting the involvement of L-type calcium channels in ALS pathogenesis.”
Similar compounds might help people with ALS, Armstrong suggested. However, he cautioned that high calcium levels can be toxic to neurons. In fact, some scientists are pursuing calcium channel blockers, not openers, to treat Parkinson’s (see ARF related news story on Kang et al., 2012; ARF related news story on Guzman et al., 2010). For people in the end stages of ALS, boosting calcium influx might make things worse, Armstrong suggested. He envisions a peripheral treatment targeting neuromuscular junctions early in disease to preserve those synapses as long as possible. A drug that does not cross the blood-brain barrier would be ideal, he said, because it could reach the junctions but might not affect calcium levels very much in the nerve cell bodies.
Unfortunately, that vision would be difficult to enact today even if researchers had a compound they could try safely in humans. Plenty of neural damage occurs before symptoms of ALS manifest, and diagnosis can take months. “There are no clinical or preclinical markers of ALS,” noted Nizar Souayah, of the University of Medicine and Dentistry of New Jersey in Newark, in an e-mail to Alzforum. “Preclinical intervention right now is not realistic.” In the meantime, Armstrong is testing the channel-opening compounds in a mouse model of TDP-43 pathology.—Amber Dance.
Armstrong GA, Drapeau P. Calcium channel agonists protect against neuromuscular dysfunction in a genetic model of TDP-43 mutation in ALS. J Neurosci. 2013 Jan 23;33(4):1741-52. Abstract