In C.elegans constitutive activation of RHO-1 in cholinergic motor-neurons (N::RHO-1*) results in animals with a fast and loopy locomotion. Previous work in our group has shown that RHO-1 increases neurotransmitter (NT) release in motor neurons, by inhibiting diacylglycerol kinase (dgk-1). However, interestingly in dgk-1 null mutants changes in RHO-1 activity can still alter NT release suggesting DGK-1 independent pathway(s). To identify other targets of RHO-1 we have conducted a genetic screen for suppressors of caRHO-1 that no longer have the loopy phenotype. Animals with N::RHO-1 were chemically mutagenized and from this screen 20 strong suppressors of the loopy locomotion was identified. Three of these suppressors were subjected to whole genome sequencing to identify potential downstream targets of RHO-1. In one of the RHO-1 suppressor strains, nz110, nz110, has a missense mutation in the MHD domain of unc-31. A putative unc-31 null mutation (e928) also suppresses N::RHO-1* loopy locomotion. UNC-31 is homologous to the mammalian calcium-dependent activator protein for secretion (CAPS). It is involved in the synaptic release of dense core vesicles (DCV’s). DCV’s transport and release neuropeptides, which likely regulates locomotion. Interestingly previous unc-31 mutations are paralyzed in the presence of food whereas our unc-31 (nz110) mutants move normally, suggesting the unc-31 (nz110) mutation only effects stimulated levels of DCV release. When we initiated the screen we assumed that suppressors of N::RHO-1* loopy locomotion would also suppress the increased release of ACh. However, many of our N::RHO-1* suppressors resulted in wild type locomotion but did not decrease ACh release. Our current model is that increased ACh release is necessary but not sufficient for loopy locomotion. We believe that an additional RHO-1 regulated signal from the cholinergic motorneurons is required for loopy locomotion. Our genetic screen suggests that the additional signal is released from DCVs, possibly a neuropeptide that acts on the muscle or other neurons. Currently, Muna is determining the site of action of UNC-31 in RHO-1 signalling and aim to test whether changes in RHO-1 activity alter levels of neurotransmitter release and if so how.
Clara has recently joined the Nurrish lab and is investigating a novel mutation, nz90, that has defects in synaptic transmission. Mapping and rescue experiments suggest that nz90 maps to a group of carboxylesterases that are related to neuroligins. Neuroligins are important for the correct functioning of mammalian synapses and defects in human neuroligins have been implicated in autism. Currently Clara is identifying the nz90 DNA mutation and determining the site of action of the carboxylases in synaptic function.
A key aim of the Nurrish group is to understand the intracellular signaling pathways that exist between extracellular neuromodulators (such as serotonin and dopamine) and the regulation of the release of neurotransmitters (such as acetylcholine). The amount of neurotransmitter released is one of the key factors in determining synaptic signal strength with synaptic signal strength known to be important in memory and learning and also addiction. The nematode worm C. elegans is therefore a good model system for studying the regulation of neuronal activity as it has only 302 neurons and 7,000 synapses compared with the vast numbers in mammals. Studies on serotonin regulation of acetylcholine release have identified the Gq alpha trimeric G-protein (EGL-30) and the RhoA GTPase (RHO-1) as important regulators of neurotransmitter release. An EMS screen for suppression of constitutively active neuronal RHO-1 generated several mutants. Whole genome sequencing was then used to identify the suppressors of neuronal RHO-1. Kimberley is studying the RHO-1 suppressor nz99, which is a mutation in the dopamine reuptake transporter DAT-1. She is currently testing if changes in extracellular dopamine alter RHO-1 activity and identifying the site of action of DAT-1 for its role in RHO-1 neuronal activity.