Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Neurosciences

Language

English (en)

Date of Award

Summer 9-1-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Paul T Kotzbauer

Abstract

The Role of Sleep in Memory Consolidation with an Induced Sleep-like State in Drosophila

Introduction: Sleep plays an important role in memory consolidation. However, the underlying mechanism remains elusive. The importance of sleep in the memory consolidation process has been demonstrated by sleep deprivation studies in which sleep loss corresponded to poor performance on memory tasks. A sleep induction approach, that allows us to study sleep-mediated changes in the presence of sleep, provides further understanding of sleep's role in the memory consolidation process.

Method: We first developed a pharmacological method to induce a sleep-like state and examined if the induced sleep-like state modulates memory consolidation in flies. Using sleep induction, we then evaluated if changes in duration and timing of the induced sleep cause the changes in memory outcomes to further understand the role of sleep in the memory consolidation process.

Results: Gaboxadol, an extra-synaptic GABAA receptor agonist, induced a sleep-like state that meets all the behavioral criteria of a spontaneous sleep-like state in flies. The induced sleep-like state following Gaboxadol treatment facilitated the formation of long-term memory in Wild-type flies. Inducing a sleep-like state in different strains of flies (ex. a series of memory mutants) and under various experimental conditions (ex. following sleep deprivation) revealed the beneficial role of induced sleep in memory processes, as supported by the observations that an increased sleep-like state rescued memory deficits in memory mutants and may compensate memory impairment caused by sleep-deprivation. By altering the duration and timing of sleep induction following the training protocol that is only able to induce a short-term memory by itself, we observed that a short-term memory was consolidated into a long-term memory even at when a sleep-like state was induced for a short period of time (<4hrs) or induced at later time (>24hrs) following the training.

Discussion: Incorporating Gaboxadol into food is a novel method to increase a sleep-like state that was behaviorally and functionally equivalent to a spontaneous sleep-like state in flies. The ability of an induced sleep to sustain the formation of long-term memory following interference periods supports the active role of sleep in the memory consolidation process. Future studies using sleep induction could complement studies from sleep deprivation and contribute to identifying genes and neuronal circuits that link sleep with memory.

The Role of Alpha-synuclein in Fatty Acid Metabolism

Introduction: Accumulation of aggregated alpha-synuclein protein is the defining pathologic feature of Parkinson's disease (PD). However, the molecular mechanisms underlying protein aggregation remain to be defined. Because the physiological function of alpha-synuclein remains unclear and previous studies have indicated a role in fatty acid metabolism and specifically acyl CoA synthesis, further studies that examine the interaction of alpha-synuclein with free fatty acids could provide a better understanding of the protein's normal function.

Method: We examined the interaction of alpha-synuclein with oleic acid, a monounsaturated free fatty acid, using a scintillation proximity assay (SPA). We characterized the regions of alpha-synuclein protein that are required for oleic acid binding using mutagenesis studies. We over-expressed alpha-synuclein in HEK293FT cells and measured its effect on the rate at which oleic acid is incorporated into phospholipids, which is determined by the rate at which oleic acid is converted to oleyl CoA by acyl CoA synthetase enzymes.

Results: We found that alpha-synuclein cooperatively binds to monomeric oleic acid. Mutations that change either amino acids 30 or 46 in the alpha-synuclein protein disrupt the alpha-synuclein-oleic acid interaction. Over-expression of WT alpha-synuclein, but not A30P alpha-synuclein, in HEK293FT cells increased the rate of oleic acid incorporation into phosphatidylcholine (PC).

Discussion: SPA is a novel approach to characterize the binding of alpha-synuclein to oleic acids. Alpha-synuclein promotes an increase in the oleic acid incorporation rate in HEK293FT cells. The absence of oleic acid binding by A30P alpha-synuclein and the corresponding absence of an effect of A30P alpha-synuclein on the incorporation rate in cells further supports the hypothesis that alpha-synuclein promotes acyl CoA synthesis by binding to free fatty acids.

DOI

https://doi.org/10.7936/K7028PKV

Comments

This work is not available online per the author’s request. For access information, please contact digital@wumail.wustl.edu or visit http://digital.wustl.edu/publish/etd-search.html URL: http://dx.doi.org/10.7936/K7028PKV

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