Date of Award
Doctor of Philosophy (PhD)
Daniel N. Cox, PhD
Victor Faundez, PhD
Sarah Pallas, PhD
Marise Parent, PhD
The Drosophila ortholog of CREB Binding Protein (dCBP) has been implicated in the pruning of sensory neuron dendrites and recent studies demonstrate that nuclear polyglutamate-induced dendritic pathologies occur, in part, by inhibiting Golgi outpost formation via a CBP-CrebA-COPII regulatory mechanism. Despite these advances, the role of dCBP in modulating dendritic development is incompletely understood. Here, we identify dCBP as a novel regulator of dendritic development that modulates the localization of Dar1, a protein known to affect dendritic growth via regulation of the microtubule severing protein Spastin and components of the Dynein complex. We discovered that dCBP is required for proper proximal-distal branch order distribution, with loss of function resulting in an aberrant reduction in terminal branching in favor of a shift towards proximal interstitial branching. Conversely, dCBP overexpression severely inhibits higher order dendritic branching in Class IV (CIV) md sensory neurons. Detailed structure-function studies using domain-specific deletions of dCBP provide further insights into the specific roles of different protein domains in mediating distinct aspects of dendritic growth. Analyses of domain-specific deletions implicate the N-terminal region (ΔNZK) in regulating the mutant phenotype, whereas expression of a deletion of the C-terminal region (ΔQ) phenocopies the overexpression phenotype. To characterize dCBP-mediated transcriptional mechanisms driving dendrite arborization, we conducted RNAseq analyses focusing on those genes that fail to be transcriptionally regulated by the ΔNZK deletion. These analyses reveal a primary role for dCBP in transcriptional repression. Enriched gene clusters included phosphorylation, ubiquitination, microtubule-based processes, protein modification processes, cytoskeletal organization, and cell morphogenesis. To characterize these putative regulatory targets, we simultaneously expressed the ΔNZK deletion construct in combination with gene-specific knockdown. These analyses revealed that disruptions of Arp53D, CG12620, CG31391, CG16716, and α-actinin 3 partially rescue aspects of morphological defects that are caused by expression of the ΔNZK construct. Combined with cytoskeletal imaging, our results suggest that dCBP function includes transcriptional repression of genes that may otherwise over-stabilize both actin and microtubule components thereby contributing to cytoskeletal dynamics required for dendritic growth. Collectively, these analyses identified transcriptional and post-translational regulatory mechanisms by which dCBP functions to direct the specification of distinct neuronal morphologies.
Clark, Sarah Glenn, "CREB Binding Protein Exerts Transcriptional and Post-translational Regulatory Effects on Dendritic Arborization in Drosophila Sensory Neurons." Dissertation, Georgia State University, 2018.
Available for download on Friday, April 19, 2019