Date of Award

January 2016

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biomedical Sciences

First Advisor

Lucia Carvelli

Abstract

Dopamine is a neurotransmitter which belongs to the catecholamine and phenethylamine families of organic compounds, and plays an important role in the regulation of reward, movement, attention, behavior and cognition. Dopamine neurotransmission is marked by the initiation of its release from the pre-synaptic neurons and the signal is terminated by re-uptake of dopamine from the synapse. The synaptic concentration of dopamine and therefore, the level dopamine receptor stimulation is regulated to an extent by the activity of the dopamine transporter (DAT). DAT is responsible for the uptake of dopamine back into the presynaptic neuron from the synapse. DAT is known to play a critical role in certain pharmacological or pathological conditions. For example, DAT is one of the major targets of several psychostimulant drugs like cocaine and amphetamine. Cocaine competitively inhibits dopamine uptake by blocking DAT which leads to increased dopamine in the synapse, one of the initial steps that promote addiction. DAT is also a major target of amphetamine (AMPH), another powerful stimulating drug. AMPH is used regularly in the treatment of neurodevelopment disorders such as attention deficit hyperactivity disorder (ADHD) but despite its role as an effective medication in such disorders, AMPH is more commonly known for its psychostimulant and addictive properties as a drug of abuse.

AMPH induces its rewarding and addictive properties by acting as a substrate of vesicular monoamine transporter (VMAT) and plasma membrane monoamine (dopamine, norepinephrine, and serotonin) transporters. AMPH is similar in structure to monoamines like dopamine, norepinephrine and therefore can bind and enter the presynaptic neurons via the transporters. Once in the neuron, AMPH causes an elevation in extracellular monoamine levels by inducing vesicular depletion of monoamines and also by promoting reverse transport (efflux) of monoamines through plasma membrane transporters. Several groups have shown that acute AMPH treatments alter the function and the number of DAT on the cell membrane but the long-term effects of chronic AMPH exposure have still not been clearly identified. Previous data from our laboratory suggested that parental AMPH exposure reduces dopamine uptake in C. elegans dopaminergic neurons isolated from progeny.Â

In order to determine if the long-term effects caused by AMPH in native C. elegans cultured neurons is reproducible in human DAT (hDAT) expressing cells, we carried out our initial investigation in LLC-PK1 porcine kidney epithelial cells stably transfected with hDAT. Cells were treated with AMPH and then analyzed for dopamine uptake, after the cells had undergone one, two or three round of division. Results from the experiments showed a significant decrease in dopamine uptake compared to untreated cells. This suggests that changes caused by AMPH were conserved up to three cell divisions. These data led us to hypothesize that long term reduction in dopamine reuptake, as a result of chronic AMPH treatment, is caused by down-regulation of DAT.

In our next approach we investigated the long term effect of AMPH in the SH-SY5Y human neuroblastoma cell lines. Previous studies have shown that the SH-SY5Y cells can be chemically differentiated into more mature neuron-like phenotype by treatment with retinoic acid (RA). Thus, we pre-treated cells with AMPH and allowed cells to cross one or more cell divisions before treating them with RA. Dopamine uptake assays revealed a significant decrease in dopamine re-accumulation in AMPH-treated cells with respect to control. Results from these experiments also revealed NET-mediated dopamine uptake in SH-SY5Y cells. In fact, desipramine, a specific NET inhibitor, totally blocked the dopamine uptake whereas GBR12909, which is a specific DAT inhibitor, did not show inhibition. Taken together, these results suggest that parental AMPH treatment down regulates the expression or activity of catecholamine transporters such as DAT and NET in daughter cells.

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