Screening for drugs at the sub-cellular level may enhance drug discovery and development for degenerative disease. The nematode C. elegans has potential for drug screening at the sub-cellular level and in vivo as it is transparent at all stages of development and can be grown in multi-well plates. To develop drug screening at the sub-cellular level in nematodes, we took advantage of the Plate Runner HD (Trophos, France), a 96/384-well device that collects fluorescence at resolutions ranging from 1024x1024 (1 px is 7.4 mm) to 8192x8192 (1 px is 1 mm). This device has high depth-of-field (about 40 mm at resolution of 7.4 mm; 8 mm at resolution of 1 mm), thus allowing fluorescent signals to be quantified from whole animals after paralysis. This device also has a wide-field objective that allows a single image of the whole well to be acquired at once.
We developed a drug screening assay at the sub-cellular level to search for compounds that may protect from the early-stage cytotoxicity of mutant PABPN1, the oculopharyngeal muscular dystrophy (OPMD) protein. Transgenic nematodes co-expressing nuclear GFP and mutant PABPN1 (PABPNA-A13) in body wall muscles show defective motility, a phenotype that is accompanied by a progressive loss of nuclear GFP signals. These phenotypes are aggravated by sirtuin (
sir-2.1/SIRT1) activation and ameliorated by
sir-2.1 inhibition, and they can be also manipulated by pharmacological means [1,2]. The loss of GFP nuclei in mutant PABPN1-A13 animals provide an easily-assayable phenotype to screen for drugs that may rescue mutant PABPN1 toxicity. Results from screening greater than 2000 compounds indicated that our screen was robust and selective. As part of a drug discovery program on neuromuscular diseases, we are also exploring the development of a drug screening assay at the sub-cellular level to search for compounds that protect from defective axonal transport. To this end, we generated transgenic nematodes that express extrachromosomal arrays encoding a fluorescent pre-synaptic reporter in GABAergic motor neurons and that carry a temperature-sensitive loss-of-function allele in a gene encoding an axon motor gene. Preliminary results suggest that, at the restrictive temperature, this mutant allele induces a significant change in the expression pattern of pre-synaptic signals. This effect is unrelated to a change in transgene expression and can be quantified by the Plate Runner HD after paralysis of the animals in the 96-well plate. Results from the two approaches abovementioned will be presented and discussed.
[1] Catoire, H. et al. (2008) Hum Mol Genet 17, 2108-2117. [2] Pasco, M.Y. et al. (2010) J Med Chem 53, 1407-1411.