Basic NeuroscienceA fluorescent-based assay for live cell, spatially resolved assessment of vesicular monoamine transporter 2-mediated neurotransmitter transport
Highlights
► We report a method for spatially resolved analysis of VMAT2 mediated uptake in live cells. ► Uptake of the fluorescent substrate was measured in HEK293 cell lines stable expressing DAT and mCherry-VMAT2. ► Uptake into the mCherry positive compartment is sensitive to tetrabenazine. ► VMAT2 activity can be quantified by analyzing confocal images. ► We have developed an assay amenable to high content imaging that is low cost, uses no animals, and is flexible compared to radioactive assays.
Introduction
The vesicular monoamine transport 2 (VMAT2; Slc18a2) is predominantly localized to the central nervous system in monoaminergic brain regions, where it packages free monoamines (dopamine, serotonin, norepinephrine, epinephrine, and histamine) in the cytosol into small synaptic and dense core vesicles (Nirenberg et al., 1998). Proper packaging of these monoamines, in particular dopamine (DA), is critical to the function and survival of these neurons. Cytosolic DA is neurotoxic but this toxicity must be balanced with the need for DA to facilitate essential behavioral functions like motor movements, learning, and the acquisition of natural rewards. Thus, greater than 90% of intracellular DA is sequestered into vesicles, preventing its cytosolic accumulation and subsequent transformation to neurotoxic species (Eisenhofer et al., 2004). The critical role of vesicular storage of DA and the effects of both pharmacological and genetic disruption has been extensively reviewed (Caudle et al., 2008, Guillot and Miller, 2009, Sulzer et al., 2005). Our laboratory has found that VMAT2-deficient mice undergo progressive degeneration of monoaminergic brain regions (the substantia nigra, locus coeruleus, and dorsal raphe) and exhibit motor and non-motor symptoms similar to those seen in Parkinson's disease (Caudle et al., 2007, Taylor et al., 2009, Taylor et al., 2011). Disruption of vesicular storage is also implicated in drug abuse (Eiden and Weihe, 2011, Sulzer, 2011). Additionally, the VMAT2-specific inhibitor tetrabenazine (TBZ) is used to treat Huntington's disease and other hyperkinetic disorders (Ondo et al., 2002, Paleacu et al., 2004). Environmental toxicants, including a variety of pesticides, organochlorine compounds, and brominated flame retardants also disrupt vesicular packaging of dopamine by VMAT2 (Bemis and Seegal, 2004, Caudle et al., 2006, Chaudhry et al., 2008, Fonnum and Mariussen, 2009, Hatcher et al., 2007, Hatcher et al., 2008, Richardson and Miller, 2004, Richardson et al., 2005). Taken together, these studies demonstrate the proper regulation of vesicular storage of monoamines is critical to the health and function of these neurons. Furthermore, the weight of evidence indicates that VMAT2 is a target of environmental contaminants and other man-made chemicals and that its study is an issue of importance to public health.
Currently, radioactive uptake assays of 3H-DA in synaptic vesicles isolated from rat or mouse brain are often used to directly assess VMAT2 function. Vesicles can be prepared from animals treated with various drugs or toxicants to determine in vivo effects (Caudle et al., 2007, Chu et al., 2010, Guillot et al., 2008, Hatcher et al., 2008, Volz et al., 2009). Alternatively, isolated vesicles can be treated directly to determine the pharmacokinetics, such as IC50 or Ki, of a compound. While this is a powerful technique to determine the actions of compounds at VMAT2, these experiments require a large amount of tissue material that necessitates the use of many animals. The amount of material obtained also limits the number of doses and time points that can be assessed in each experiment. Two alternate techniques in cell lines bypass these limitations. In the first method, cells are treated with detergent to permeabilize the plasma membrane while leaving the vesicle membrane and machinery intact (Erickson et al., 1996). The second method involves isolation of a post-nuclear fraction from cell lines stably expressing VMAT2; tetrabenazine (TBZ, a specific VMAT2 inhibitor)-sensitive uptake can be detected in this fraction (Bellocchio et al., 2000, Parra et al., 2008). However, none of these vesicular uptake assays provides an understanding of the action of these compounds in a whole cell. They do not allow for assessment of access to the vesicle, combined action at plasma membrane and vesicular transporters or indirect mechanisms of regulation. For example, a compound that affects VMAT2 function in isolated vesicles may not be able to cross the plasma membrane; such a compound would have no effect on VMAT2 function in a whole cell. In addition, these methods are not amenable to a high throughput format primarily due to their use of radioactivity. Furthermore, adhering an isolated vesicle fraction to a plate is technically challenging and the partial permeabilization method requires multiple washing steps. Therefore, we sought to develop a method that could measure vesicular uptake in whole cells and be suitable for adaptation to a high-throughput format.
We modified the Neurotransmitter Uptake Assay (Molecular Devices), which is marketed to measure uptake through the plasma membrane monoamine transporters. This assay consists of a proprietary fluorescent compound that is transported into cells by the plasma membrane monoamine transporters and a non-permeable masking dye to quench fluorescence in the media. Here, we demonstrate that this dye is transported by VMAT2 and can be adapted to measure uptake in real time in intact cells.
Section snippets
Cell culture
HEK293 cells (ATCC) and HEK293 lines stably expressing hDAT, hVMAT2, mCherry-hVMAT2, hDAT and hVMAT2, or hDAT and mCherry-VMAT2 constructs were cultured at 37 °C and 5% CO2 in DMEM with 10% FBS. All constructs were made in pcDNA3.1 (Life Technologies). hDAT and mCherry-hVMAT2 expressing constructs contained a neomycin or zeocin resistance gene, respectively. Plasmids were transfected into HEK293 cells with Lipofectamine 2000. Stable cell lines were generated by repetitive rounds of limiting
The fluorescent substrate is transported by DAT and VMAT2
The fluorescent reporter dye is marketed as a substrate of the plasma membrane monoamine transporters (the dopamine, norepinephrine, and serotonin transporters; DAT, NET, and SERT). Since this dye is an analog of MPP+, which is transported by DAT and VMAT2, we hypothesized that this dye is also a VMAT2 substrate. To test this, we generated HEK293 cell lines stably expressing human DAT, human VMAT2 or both (HEK-DAT, HEK-VMAT2, or HEK-DAT/VMAT2, respectively). These cells were incubated with the
A fluorescent, spatially resolved assay for vesicular packaging in live cells
Radioactive neurotransmitter uptake by isolated synaptic vesicles has produced many insights into the pharmacology of VMAT2. However, the amount of brain tissue required limits the number of time points, doses and compounds that can be measured. The fluorescent-based assay described here overcomes this limitation because cell lines are a practically unlimited resource. Assay conditions are not limited by the amount of material required as cell numbers adequate for multiple replicates,
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