This revised F30 application is to provide support for the completion of my DDS/PhD training in the Oral & Craniofacial Sciences Program at UCSF. I am currently enrolled in Year 3 of the program, and I have entered the DDS/PhD integrated curriculum after successfully passing my qualifying exam and publishing a first author manuscript describing the initial characterization of our Wdr72-/- mouse and a novel WDR72 mutation in individuals with a hypomaturation enamel phenotype of Amelogenesis Imperfecta. I will continue my integrated DDS/PhD training by focusing on understanding how WDR72 regulates intracellular processes in response to the environment to promote enamel mineralization. Mutations in WDR72 result in Amelogenesis Imperfecta (AI), though the mechanism is unknown. Patients with mutated copies of the WDR72 gene have severely hypomineralized enamel, and our Wdr72-/- mice show severely hypomineralized enamel with evidence of reduced vesicle markers at the maturation stage. Both human and mouse tooth phenotypes, as well as mRNA and protein expression studies, suggest a role for WDR72 as an intracellular regulator during the maturation stage of enamel formation. Our molecular modeling has suggested a role for WDR72 as a vesicle coat protein, possibly regulated by the high Ca2+ environments experienced during enamel maturation. These possibilities are supported by my published and preliminary studies, which show WDR72 co-localizing with an endosome H+/Cl- exchange protein, CLC5, that works in concert with vacuolar H+-ATPase (v-ATPase) to acidify vesicles. Additional preliminary data suggest direct interaction between WDR72 and Ca2+, showing WDR72 with a conserved Ca2+-binding motif, and my cell culture work suggests that WDR72 forms vesicle-like assemblies in high Ca2+ environments. Moreover, WDR72's paralog, WDR7, regulates transport of v-ATPase needed for proper endocytosis and acidification, in addition to functioning as a scaffold in Ca2+-dependent vesicle mobilization at neural synapses. Based on my findings and the known functions of WDR72's paralog, I propose to investigate the central hypothesis that WDR72 forms a vesicle coat, a process mediated by Ca2+ in order to regulate enamel mineralization in maturation-stage ameloblasts. This hypothesis will be tested with the following specific aims:
Specific Aim 1 : Determine if WDR72 is a vesicle coat protein in ameloblasts involved in complete enamel mineralization.
Specific Aim 2 : Determine if Ca2+ regulates WDR72 assembly to promote vesicle acidification. Completion of this PhD project paired with my DDS training will provide me with a strong foundation for my future as a translational clinician-scientist and will direct our knowledge of WDR72 function in vesicle trafficking and enamel development to drive scientific understanding of the dynamic intracellular processes underlying biomineralization.
Mutations in WDR72 represent one form of Amelogenesis imperfecta (AI), a genetic disorder that manifests as a severe tooth enamel defect. The research strategy proposed in this application aims to investigate WDR72 function during tooth formation, specifically in vesicle-mediated control of enamel mineralization. Funding of this grant application will allow me to continue my DDS/PhD training and will provide me with the tools to succeed in a career as a translational craniofacial scientist.