Gap junctions play essential roles in many biological processes, such as embryo development, cell differentiation, cell growth, metabolic coordination of avascular organs, and neural development, and misregulation of gap junctions has been linked to many diseases. However, the molecular mechanisms underlying gap junction formation and regulation are still largely unknown. Using C. elegans PLM neurons as a model, we found that functional GFP-tagged innexins form plaque structures that represent the location of gap junctions in vivo. We then carried out an unbiased genetic screen using transgenes expressing GFP-tagged innexins and uncovered 12 mutants with 3 types of defects in gap junctions. Based on mutants isolated from this genetic screen, we outline 3 aims in this proposal to study mechanisms for gap junction formation, turnover, and elimination. In the preliminary studies we discovered the previously unknown function of CED- 10/Rac and MEC-15/F-box/WD repeat-containing protein in regulating gap junction formation, and in Specific Aim 1 we outline a plan to investigate the function and regulation of CED-10 and MEC-15 in gap junction formation. Regulation of gap junction turnover plays an important role in gap junction functions. In our previous study, we revealed that the C. elegans ankyrin protein UNC-44 and CRMP UNC-33 regulate gap junction turnover. However, neither unc-44 nor unc-33 mutants have completely penetrant phenotypes, suggesting that other pathways are involved in regulating gap junction turnover. In this proposal we present evidence to show that the C. elegans titin UNC-22 functions in parallel with the UNC-44/UNC-33 pathway and is likely regulated by microtubules to modulate gap junction turnover.
In Aim 2 we propose to study the regulatory mechanisms of UNC-22/titin and microtubules and their crosstalk with the UNC-44/UNC-33 pathway in gap junction turnover. During neuronal development gap junction channels are expressed on the membrane before gap junction formation, and little is known about the distribution and function of gap junction channels at this stage. We show that UNC-9/innexin forms puncta along the axon in PLM neurons before the formation of gap junctions, and these transient clusters of gap junction channels are eliminated by the autophagy pathway when neurons form gap junctions.
In Aim 3 we outline a plan to address the function of these transient clusters of gap junction channels and the autophagy pathway in regulating neuronal development. Completion of this proposal will lead to the discovery of novel mechanisms of gap junction formation and regulation, the establishment of C. elegans PLM neurons as a powerful model to study gap junctions, and the generation of new tools for further studies. Given that many neural disorders are associated with defects in gap junctions, this project will likely aid in the understanding of brain development and functions in both physiological and pathological conditions.

Public Health Relevance

Gap junctions play critical roles in brain development and function, and disruption of gap junctions has been linked to many birth conditions and neurological diseases. In this proposal, we take advantage of the model organism Caenorhabditis elegans to investigate novel mechanisms of gap junction formation and regulation. Completion of our studies will pave the way to fully understand gap junctions and likely provide new drug targets for potential clinical studies of neurodevelopmental and neurodegenerative disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS105638-03
Application #
9957134
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Leenders, Miriam
Project Start
2018-09-30
Project End
2023-06-30
Budget Start
2020-07-01
Budget End
2021-06-30
Support Year
3
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Duke University
Department
Genetics
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705