Development of a screening assay for vesicular dopamine transport Público
Wilson, Bethany Kaye (2016)
Abstract
The vesicular monoamine transporter 2 (VMAT2; Slc18a2) packages monoamines into vesicles for subsequent release and neurotransmission and is critical for sequestering both exogenous and endogenous toxicants away from their sites of action inside the neuron. We have previously found that genetic reduction of VMAT2 in a mouse model reproduces many of the features of Parkinson's disease and shows increased neurotoxic vulnerability. Other studies demonstrate that environmental chemicals can inhibit VMAT2 and that VMAT2 is dysfunctional in Parkinson's disease. Conversely, we have found that genetic increase of VMAT2 expression in mice results in elevated levels of dopamine in the brain, increased vesicular packaging of dopamine, increased dopamine release, and resistance to toxic insult. Thus, VMAT2 appears to be a valid target of interest for pharmacological treatment of Parkinson's disease and other monoamine-related disorders, as well as a measure of the impact of environmental exposures. We have developed and optimized an assay suitable for the pharmacological and toxicological screening of compounds, which positively or negatively affect vesicular dopamine transport. This assay is designed to evaluate modulators of VMAT2 function, but will also identify compounds that affect the expression or trafficking of VMAT2 and the function of other vesicular proteins; specific effects can be elucidated through secondary screens. The assay is based on the new fluorescent false neurotransmitter dye FFN206, which is selectively transported by VMAT2. The assay is tetrabenazine-sensitive and amenable to the 96-well format. Furthermore, we have optimized the assay for multiple conditions including cell line, plating density, plating technique, plate material and coating, well size, reaction volume, experimental media, negative control, reagent handling and storage conditions, test compound concentration and incubation time, FFN concentration and incubation time, FFN incubation temperature, wash temperature and volume, reading volume, plate reader type and temperature, and plate reading dimensions. In so doing, we have achieved a z-factor of 0.93, which is suitable for high throughput screens, and scalable to robotic adaptation and miniaturization in a 384 or 1536 well format.
Table of Contents
Introduction. 1
Materials and Methods. 11
Results. 14
Discussion. 19
Figure Legends. 24
References. 30
Figures. 38
About this Master's Thesis
| School | |
|---|---|
| Department | |
| Subfield / Discipline | |
| Degree | |
| Submission | |
| Language |
|
| Research Field | |
| Palabra Clave | |
| Committee Chair / Thesis Advisor | |
| Committee Members |
Primary PDF
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
Development of a screening assay for vesicular dopamine transport () | 2018-08-28 14:48:41 -0400 |
|
Supplemental Files
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
Thesis Figures 2016.04.04.pdf () | 2018-08-28 14:48:59 -0400 |
|