Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Neurosciences

Language

English (en)

Date of Award

Winter 1-1-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Paul H Taghert

Abstract

Daily rhythms in physiology are conserved throughout evolution. The molecular oscillations that underlie rhythms have been well characterized and are required in a specific set of pacemaker cells in the Drosophila brain for locomotor rhythms. These cells are divided into distinct subgroups and evidence suggests that subgroups of pacemaker cells may control distinct components of circadian behavior although these assignments are not rigid, and depend critically upon environmental conditions. Plasticity in the neural circuits controlling circadian behavior allow the animal flexibility in responding to fluctuations in environmental inputs, for example changing day length over seasons.

Intercellular communication between diverse clock cells is key for normal circadian behavior. This communication is mediated through the actions of the neuropeptide PDF: Pigment Dispersing Factor). Although increasing evidence suggests that downstream effectors differ between subgroups the molecular mechanisms of these differential responses remain unknown. Defining these mechanisms are of fundamental importance to understanding the widespread and robust synchronizing actions of PDF across the circadian neuronal circuit. This thesis explores cell signaling components associated with the PDF receptor in two clock cell subgroups; small ventro-lateral neurons: small LNv) and dorsolateral neurons: LNd).

Using a genetically encoded sensor to measure changes in cAMP in the living Drosophila brain, I report that PDF receptor preferentially couples to specific signaling components. These groupings vary in composition depending upon receptor identity; small LNv cells express Gsalpha coupled receptors for the peptide DH31, PDF as well as dopamine. However, these three receptors show different coupling at the level of adenylate cyclase. PDF receptor is preferentially coupled to AC3 in the small LNv cells, however DH31 and dopamine responses are unaffected by manipulations of AC3. Surprisingly, another subgroup of PDF receptor expressing clock cells, LNd cells, do not rely upon AC3 to mediate PDF responsiveness. Instead, the PDF response in these cells is reduced after knockdown of AC78C. A class of scaffolding proteins called AKAPs: A-kinase anchoring proteins) are implicated in PDF responses in both subgroups presumably by allowing signaling complexes to form efficiently. My work suggests that downstream components form "signalosomes" in specific pacemaker cells in the Drosophila brain.

I report that small LNvs show diurnal variation in sensitivity to PDF peptide. My studies support a model in which modifications that affect localization of signaling components may play important roles in changes in receptor signaling. Previous work performed in the mammalian olfactory epithelium suggests that specific glycan modifications are required for AC3 enzymatic activity and that these modifications rely upon a specific glycosyltransferase: beta-3GnT2). I report that knockdown of CG30036, the Drosophila homolog of beta-3GnT2, reduces PDF responses in small LNv cells and causes circadian disruption partially consistent with AC3 defects. Glycosylation sites are conserved among transmembrane ACs and I identified a separate glycosylstransferase: CG33145) that, when knocked down, reduces LNd PDF responses. These findings are consistent with a model of PDF receptor complexes that differ in their composition in different subgroups of pacemaker cells. This model provides a possible mechanism to explain the differences in downstream PDF responses between clock cell subgroups.

Additionally, I provide evidence that two forms of the Diuretic Hormone Peptide: DH31 and DH81) show differential preferences for two GPCRs: CG4395 and CG17415) in vivo. These two receptors are members of the CGRP family of peptide receptors, the third member of which is PDF receptor. DH81 is an 81 amino acid peptide and this greatly extends the length of known bioactive peptides, for which in vivo evidence is critical. Additionally, this finding suggests that, although they are encoded by the same gene, that peptide processing may functionally encode multiple neurotransmitter pathways. This finding may provide a mechanism for an additional level of signaling complexity within the Drosophila brain.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7SJ1HMJ

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