Rho GTPase Signaling Modulates Neurotransmission in Caenorhabditis elegans

Rho family GTPases act as molecular switches in the regulation of diverse cellular functions, including cell division, gene transcription and neurotransmission. In the model organism Caenorhabditis elegans, the Kalirin and Trio ortholog UNC-73 contains two RhoGEF domains that specifically activate either Rac or Rho GTPases, respectively. The RhoGEF1 domain and the Rac pathway are required for axon guidance in neuronal development, while the RhoGEF2 domain is involved in the control of locomotion, possibly through the modulation of neurotransmission. The unc-73 gene is expressed as multiple isoforms that contain either one or both RhoGEF domains. This study focuses on defining the function of the UNC-73 RhoGEF2 containing isoforms and the Rho signaling pathway in the adult nervous system of C. elegans.Animals with mutations in the unc-73 RhoGEF2 domain move more slowly than wild-type animals and this locomotory defect can be rescued by pan-neuronal expression of the UNC-73E isoform, which contains only the RhoGEF2 domain. In addition, unc-73 RhoGEF2 mutants are resistant to the cholinesterase inhibitor aldicarb and are hypersensitive to the cholinergic agonist levamisole, without any obvious changes in synaptic structure based on the localization of fluorescence-tagged synaptobrevin. These results suggest that the UNC-73 RhoGEF2 isoforms may modulate synaptic strength and cholinergic signaling at the level of the neuromuscular junction. Neuropeptide signaling is also affected in UNC-73 RhoGEF2 mutants. RhoGEF2 mutants expressing the YFP-tagged neuropeptide NLP-21 in cholinergic motorneurons exhibit decreased axonal, but not somal, YFP fluorescence compared to wild-type animals, suggesting NLP-21::YFP packaging into vesicles or transport to the axons is inhibited. YFP fluorescence in the coelomocytes, an indicator of neuropeptide release, is also decreased in these animals. These results suggest the UNC-73 RhoGEF2 isoforms may play a neuromodulatory role in the regulation of locomotion, perhaps by regulating dense core vesicle (DCV)-mediated neuropeptide signaling at the level of axonal transport or neuropeptide packaging. The lethargic locomotory and aldicarb resistant phenotypes of unc-73 RhoGEF2 mutants were bypassed in animals with constitutively active (CA) Gas signaling. Gas(CA) expression is required in both muscles and neurons to rescue unc-73 locomotory phenotypes in RhoGEF2 mutants, as the expression in either muscle or neurons alone failed to yield substantial rescue. unc-73 RhoGEF2 mutants exhibited phenotypes similar to rab-2 and unc-31 mutants, both of which encode proteins that are involved in DCV-mediated signaling. Gas(CA) signaling also rescues unc-31 and rab-2 locomotory defects, supporting our hypothesis that UNC-73 RhoGEF2 signaling may modulate DCV-mediated neurotransmission. Together, these results indicate UNC-73 RhoGEF2 isoforms may regulate cholinergic signaling indirectly by modulating neuropeptide signaling and that the Gas pathway acts downstream of, or in parallel to, the neuropeptide signaling pathway.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Biology (Cell-Molecular Biology)

Grantor dc:publisher

University of Toledo

Year dc:date

2013

Author dc:creator

• Hu, Shuang

Contributors dc:contributor

• Steven, Robert

• Biology
• Neurosciences

Statement dc:rights

• unrestricted
• This thesis or dissertation is protected by copyright: some rights reserved. It is licensed for use under a Creative Commons license. Specific terms and permissions are available from this document's record in the OhioLINK ETD Center.

Language dc:language

English

Repository record dc:identifier

http://rave.ohiolink.edu/etdc/view?acc_num=toledo1365182177

OAI identifier oai:identifier

oai:etd.ohiolink.edu:toledo1365182177