Microtubules constitute a major component of the neuronal cytoskeleton, and tight regulation of microtubule dynamics is critical in establishing and maintaining proper cellular structure and function. As such, microtubule regulation is central to neuronal differentiation, as well as recovery from neuronal injury. Previously, our lab identified a protein in Lymnaea stagnalis (pond snail) called caltubin that is required for neurite outgrowth and regeneration in snails, and that is sufficient to increase neurite outgrowth and limit retraction of lesioned neurites when introduced into mammalian central neurons by plasmid transfection (Nejatbakhsh et al. 2011). Mechanistically, it was established that caltubin contains 4 putative EF-hand calcium binding domains and associates with tubulin. In this thesis project, a cell penetrating version of caltubin was created for more efficient delivery of caltubin into cells in vitro, with the added capability of being able to efficiently deliver caltubin in vivo. Functionally, this protein promoted neurite outgrowth and short term regeneration in mammalian central neurons in culture, while increasing the stability of microtubules. Mechanistically, it was determined that caltubin (1) promotes the assembly of microtubules directly, and (2) interferes with the function of tubulin tyrosine ligase â an enzyme that catalyzes the retyrosination of tubulin â and thus may also indirectly affect tyrosination-sensitive mechanisms of microtubule regulation. Furthermore, it was determined that calcium: (1) binds to caltubin, (2) changes its conformation, (3) increases its binding to tubulin, and (4) enhances its effect on neurite outgrowth. Finally, specific caltubin-tubulin binding domains have been identified, as well as the caltubin functional domain that promotes neurite outgrowth. Taken together, it has been established that caltubin functions as an effective and regulatable enhancer of microtubule assembly and thus could potentially have therapeutic value as a neuroprotectant or enhancer of neuronal regeneration.Ph.D.2018-11-30 00:00:0
