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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - LEVADAPT (Identification of new regulators of Acetylcholine receptor using the nematode Caenorhabditis elegans)

Teaser

Summary

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that have been highly conserved throughout evolution and play key roles in numerous physiological and pathological processes in humans. In the central nervous system, nAChRs have important roles in diverse processes including learning, memory and attention. Alterations of nAChR-related signalling contribute to neurodegenerative disorders such as Alzheimerâ€™s or Parkinsonâ€™s disease. In the peripheral nervous system, nAChRs are responsible for the fast-excitatory neurotransmission at neuromuscular junctions. At the neuromuscular junction, AChRs are localized at the surface of the muscles where they are activated by the release of acetylcholine from motoneuron. Activation of AChRs eventually trigger muscle contraction and then malfunction of AChRs lead to severe neuromuscular diseases such as myasthenia gravis or congenital myasthenic syndrome.
The free-living nematode worm Caenorhabditis elegans is a powerful model organism to study neuromuscular system and AChR function. In C. elegans, as in mammals, acetylcholine is the neurotransmitter used to trigger muscle contraction. A subclass of AChR present at the neuromuscluar junction has been well studied due to its sensitivity to levamisole, a nematode-specific agonist . Prolonged exposure to levamisole causes hyperactivation of AChR and then hypercontraction of C. elegans muscles leading to paralysis and eventually death of worms. However, mutations in genes regulating AChR biosynthesis, activity or clustering modify the response of the worms to levamisole.
As wild-type worms, the mutant worms initially paralysed when exposed to levamisole but after several hours they remain hypercontracted and start to move again. At the molecular level, this hypercontraction of adapting worms is characterized by a sustained elevated level of calcium in the muscle which is not observed in wild-type animals.
This project aimed to characterize the mechanisms that allow adapting worms to overcome the stress induced by sustained hypercontraction of muscle cells and to recover locomotion in a such situation. More generally, the goal is to study the mechanisms underlying the adaptation of striated muscle cells to the stress triggered by muscle hyperactivity.

Work performed

In this project, we used two differents strategies to try to identify proteins involved in the process of adaptation to levamisole: a unbiased strategy and a candidate-based approach.
In the first one, we used the results from a genetic screen previously conducted in the lab in order to identify new components involved in the adaptation to levamisole. In this screen, worms expressing the AChR subunit UNC-29 tagged with RFP was mutagenized with a very powerful mutagen inducing random mutations into the genome. All mutagenized worms were screened on their ability to adapt to high concentration of levamisole.
For all the adapting strains, the organization of AChR at the muscle cell membrane was analysed by fluorescence microscopy by looking at UNC-29-RFP. Strains where AChRs were absent or declustered were discarded and only strains with a pattern of fluorescence similar to the one of the non-mutagenized strain were further analysed. This strategy was adopted to discard mutants with severe synaptic disorganization and it was also a way to find new genes regulating AChR-related signalling in a manner different from the proteins identified so far in the lab. This screen finally isolated 7 strains with no mutation in the already known candidates but able to adapt to levamisole. The first aim of this was to identify the mutations in these strains. By crossing the 7 strains with the reference strain, we discarded 3 strains that become only mildly able to adapt to levamisole. We then sequenced the entire genome of the 4 remaining strains. However, despite extensive work to spot the causative mutation for adaptation we were not able to find a precise mutation in the genome of these 4 strains.
We then focused on a second strategy based on a candidate-based approach. As the level of calcium remains high in the muscles of adapting worms all along levamisole exposure, we decided to focus on candidates that are dependent on calcium for their activity. We first look if the calcium-dependent phopshatase Calcineurin, tax-6 in C. elegans, is involved in the adaptation to levamisole. Preliminary experiments using worms that are mutant for tax-6 support the idea that this protein is implicated in the adaptation. However, these mutants are quite sick and have strong locomotion defects. To validate the implication of tax-6 in the response to levamisole we took advantage of the auxin-inducible degradation system which allow the rapid degradation of a protein of interest in a specific tissue at a given time. Using this technique we showed that tax-6 is required specifically in the muscle for the adaptation to levamisole.
We then investigated which pathway could be activated downstream of tax-6 in adapting worms. Our experiments suggest that the transcription factor CREB is involved in the adaption to levamisole probably through the calcineurin-dependent activation of crtc-1, a co-activator a CREB. Our results then suggest that the adaptation to levamisole requires the establishment of a transcriptional program that may reshape the muscle cell to allow it to deal with the new cellular homeostasis that represent the sustained high calcium concentration.
These results have been presented in 2 international conferences in the USA and in 2 national conferences.

Final results

This study have pave the way to the understanding of the mechanisms underlying the adaptation of striated muscle cells to the stress triggered by muscle hyperactivity. It showed how muscle cells can implemented metabolic adaptations to face a change in cellular homeostasis. The mechanisms characterized here may reflect the ones activated in humans during prolonged exercise. This plasticity of muscle cells may be impaired in some neuromuscular diseases where muscles can not deal with sustained activity. Deciphering the mechanisms allowing this muscular plasticity may then help to understand some pathological situations.