GSBS Dissertations and Theses

ORCID ID

0000-0002-8600-0364

Publication Date

2020-04-30

Document Type

Doctoral Dissertation

Academic Program

Neuroscience

Department

Neurobiology

First Thesis Advisor

Haley Melikian

Keywords

dopamine, dopamine transporter, protein kinase C, rit2, GTPase, membrane trafficking, endocytosis, striatum, drosophila, sleep, sleep consolidation

Abstract

Dopamine (DA) governs movement, sleep, reward, and cognition. The presynaptic dopamine transporter (DAT), clears released DA, controlling DA signaling and homeostasis. Genetic DAT ablation causes hyperactivity, sleep reduction, and altered psychostimulant response. DAT surface expression is dynamic; DAT constitutively internalizes and recycles to and from the plasma membrane, and acute PKC activation stimulates DAT endocytosis. Cell line experiments demonstrated that PKC-stimulated DAT endocytosis requires Ack1 inactivation and the GTPase, Rit2. How Rit2 controls PKC-dependent DAT internalization, or whether regulated DAT endocytosis impacts behavior, is unknown. Here, I present data supporting that PKC activation stimulates Rit2/DAT dissociation, mediated by the DAT N-terminus. Further, Ack1 and Rit2 function independently to facilitate PKC-stimulated DAT internalization. Moreover, PKC-stimulated DAT endocytosis was limited to ventral striatum in ex vivo slice preparations, and required Rit2. Our lab previously demonstrated that certain DA-dependent behaviors required DAergic Rit2 in mice, however whether this was due to perturbed PKC-stimulated DAT internalization, or DAT-independent Rit2 function(s) remains untested. To address this, I turned to Drosophila and its Rit2 homolog Ric. I found that Ric and dDAT proteins interact in cell lines, and that constitutively active Ric (RicQ117L) increased dDAT function in cultured cells and ex vivo whole fly brains. However, neither DAergic Ric knockdown nor RicQ117L altered overall locomotion or sleep, suggesting that these fundamental behaviors do not require DAergic Ric. Together, these results expand our understanding of intrinsic mechanisms controlling DAT endocytosis, and their impact on behavior.

DOI

10.13028/bydf-cx52

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Copyright is held by the author, with all rights reserved.

Available for download on Sunday, May 23, 2021

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