Molecular and genetic determinants of the inhibitory action of emodepside on C. elegans muscle

Abstract

The increasing resistance of parasitic nematodes to existing anthelmintics has encouraged the search for novel compounds.  Emodepside, a 24 membered cyclic depsipeptide, has been shown to act as a potent broad-spectrum anthelmintic.  Emodepside causes a fast onset of paralysis of nematodes, favouring the view that it acts on nerve and muscle systems.  The basis of this study was to further investigate the mode of action of emodepside, using the parasitic nematode  A. suum and the free-living non-parasitic model genetic organism C. elegans.

The effects of  emodepside and GABA were investigated on the rate of relaxation of A. suum muscle pre-contracted with acetylcholine (ACh) using an organ bath preparation.  The relaxing effect of GABA was more rapid than that of emodepside.  Since the GABA effect on A. suum and emodepside effects on pre-contracted muscle was investigated using chloride-free saline.  While the effect of emodepside was unaffected in chloride-free saline, the effect of GABA was converted to a very slow relaxation, or in some cases, a slight contraction.  These data suggest that the effect of emodepside is not mediated through a direct GABAergic pathway.

To provide an insight into the molecular mechanisms and signalling pathway involved in emodepside action, the model organism C. elegans was used.  Emodepside inhibits locomotory behaviour (IC₅₀ 4.1nM), and this assay was used as a screen to delineate the mechanism of action of the drug.  HC110R, a latrophilin-like receptor, was identified in H. contortus, as a possible target site for emodepside (Saeger et al., 2001).  In C. elegans two latrophilin-like genes exist; lat-1and lat-2.  Individual RNAi to lat-1 and lat-2 resulted in a significant reduction in sensitivity to emodepside.  The lat-2 (ok301) deletion strain also exhibited low level emodepside resistance.

Evidence for stimulation of vesicle release by emodepside was provided by imaging of synapses using the fluorescent dye FM4-64 (Wilson et al., 2004).  Intracellular uptake of FM4-64 results in fluorescence of synaptic boutons.  A rapid and selective loss of fluorescence was observed following application of emodepside, indicating vesicle exocytosis in the presence of emodepside.

The signalling pathway through which emodepside stimulates vesicle release was also investigated.  Egl-30 (Gα_q) and egl-8 (phospholipase-Cβ) loss-of-function mutations both resulted in decreased sensitivity to emodepside, whereas egl-30 gain-of-function mutations resulted in hypersensitivity to emodepside.

Investigation into the potential neurotransmitters that are released following emodepside stimulation, suggests that a cocktail of neurotransmitters may be released at the NMJ, including ACh and inhibitory neuropeptides.  A reduced sensitivity to emodepside was observed following RNAi for the flp-1 and flp-13 genes.

Emodepside appears to have a complex mode of anthelmintic action, involving a presynaptic latrophilin-dependent pathway and the necessity for the SLO-1 channel.

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